Instabilities of proteins: theoretical aspects, degradation products and methods for their detection

Recombinant DNA technology has led to a significant increase in the number of peptide and protein based pharmaceuticals, giving a new approach to combat poorly controlled diseases. This particular development has been reached in the last two decades. However, proteins are highly susceptible of physical and chemical degradation resulting in a decrease or complete loss of biological activities. Reasons for their physical and chemical instabilities and the methods for their examination, become a challenge for the pharmaceutical scientists for successful development of stabile protein based pharmaceuticals. The stability of protein based pharmaceuticals is significant in terms of their pharmaceutical quality and biological activity. In addition, a right choice of suitable analytical methods is needed in order to detect an early formation of degradation products or modified forms.


Inroduction
During the past two decades, peptides and proteins have become an important class of potent therapeutic drugs.They have gained significant importance in the treatment of several severe diseases including autoimmune diseases, memory impairment, hormonal disorders, organ transplantations and different cancers.Major advantages of protein drugs are both their extremely specific activity and their high tolerability (1).
While recombinant DNA technology, which is providing exciting opportunities for new pharmaceutical development and new approaches to the diagnosis, treatment and prevention of diseases, has led to a significant increase in the production of peptides and proteins for pharmaceutical purposes, this has not been matched by the number of peptide and protein based drugs available on the market.The reasons for that are multiple: proteins highly susceptibility to chemical and physical degradation, which is associated with relevant difficulties in purification, storage and delivery, multifaceted metabolic properties, variable tissue penetration and toxicity related to the stimulation of the immune or allergic reaction (2,3,4).
The structure and function of proteins is determined by their amino acid sequence which defines the peptide backbone, the primary structure.In spite of the enormous number of naturally occurring proteins, a mere of 20 amino acids construct proteins.The vast difference in the threedimensional structure and, therefore, also in protein function originates solely from a different amino acid sequence, i.e. the unique structure of a protein is determined by the chemical and physical properties of the amino acids aligned within the protein sequence.The secondary structure describes the folding or the shape of the polypeptide chains into regular, ordered structures like α-helices and β-sheets.Furthermore, areas with increased flexibility -the so-called turns or loops -are to be subsumed in this level of protein structure.The domains of the secondary structure and all noncovalent interactions such as hydrogen bonds and hydrophobic, electrostatic, or van der Waals interactions generate the intrinsic, three-dimensional arrangement of a protein, the tertiary structure.Some proteins consist of several polypeptide chains.The quaternary structure characterises the non-covalent interactions binding these chains into a single protein molecule (5).For example, haemoglobin consists of four polypeptide chains, which are associated by one Fe 2+ ion.The retention of the tertiary structure is deemed the primary requirement for the biological activity of protein molecules.However, the biochemical and structural complexity of these molecules is the reason for proteins to react sensitive to even marginal changes in their natural environment (6).
In the context of protein structure, the term stability can be defined as the tendency to maintain a native (biologically active) conformation.
As proteins and peptides continue to enter the pharmaceutical market, their stability becomes a pressing issue for the pharmaceutical scientists.Native proteins are only marginally stable and highly susceptible to degradation, both chemical and physical (7)(8)(9).
Chemical instability refers to the formation or destruction of covalent bonds, within a protein molecule, i.e. some amino acid side chains are chemically reactive, whereas others are chemically inert.It has been demonstrated that "labile" amino acid residues are susceptible to covalent modifications via bond formation or cleavage through nonenzymatic reactions, including hydrolysis, deamidation, oxidation, racemisation, β-elimination and cystine destruction/disulfide exchange (10,11).These changes alter the primary structure of the protein, and impact higher level of its structure.
The primary focus of this minireview is to present and discuss the main reasons for the most common physical and chemical instabilities and the methods for their detection.It is important to remember that every protein is unique, both physically and chemically, and therefore exhibits unique stability behaviour.

Denaturation
The loss of tertiary structure, and frequently also of secondary structure is generally referred to as denaturation of the protein.Perturbation of secondary or tertiary structure can lead to exposure of previously buried amino acid, facilitating its chemical reactivity; thereby leading to loss of its native or original characteristics.Denaturation can be caused by destabilizing agents such as excipients (reducing sugars, antioxidants, surfactants, metal ions), heat, hydrolysis by strong acid or alkali, enzymatic action, exposure to urea or other substances, or exposure to ultra violet light.Exci-pients like reducing sugars can react with protein amino groups to form schiff's bases (Maillard reaction) which can be shown by LC/MS (13).Antioxidants may contain reducing agents that will destroy disulphide bonds.Several surfactants like Tween 20 and Tween 80 can cause oxidation of aminoacid due to residual peroxides present in these materials, which can be detected by RP-HPLC (14).Many chemical reactions involving polypeptides and proteins are catalyzed by metal ions such as Zn 2+ , Cd 2+ , Pb 2+ .At high temperature (80-100°), aspargine and glutamine are susceptible to deamidation, Aspartate-Xaa peptide bonds are susceptible to hydrolysis, disulphide bonds rupture, and Xaa-Prolin peptide bonds undergo cis-trans isomerisation (where Xaa is any amino acid).High temperature can result in physical degradation due to irreversible denaturation.Residual moisture can be responsible for protein instability in the solid state.pH has also strong influence on denaturation and aggregation rate (15,16).
The folded state of proteins is connected to conformational stability, which is expressed as the free energy change ΔG during the unfolding/denaturation reaction under physiological conditions (17).The higher the ΔG value, the greater the stability of the protein.However, the reported ΔG values for proteins of 45 +/-15 kJ/mol indicate that the folding state is only marginally more stable than the denatured state (18).As a matter of fact, the conformational stability of a protein in aqueous solution tallies with only a few hydrogen bonds or ion pairs (2).Simplified, protein denaturation can be described as: The native state N exists in an equilibrium with a partially unfolded state pU.This unfolding of the native protein can be reversible, e.g. an increase of the temperature causes unfolding, which can be reversed by a subsequent temperature decrease (7).Generally, the loss of the tertiary structure implies an increase in the protein molecule's reactivity, which results in a decrease in their stability.Hydrophobic regions, which were accumulated in the core of the folded protein, are then exposed to surrounding solvents.As a consequence, side reactions can now lead to an irreversible, denatured state D. Alternatively, partially unfolded proteins may encounter irreversible aggregation (pU)n.Constantly elevated temperatures, extreme pHs, the formation of interfaces during shaking, shearing, adsorption to hydrophobic surfaces, high pressure, and denatu-rants such as urea and guanidine hydrochloride foster irreversible transitions (2,(19)(20)(21)(22)(23).

Physical instability
Physical instability of proteins denotes that these super structured molecules can undergo changes independent of any chemical modification, i.e. includes the reactions that do not involve the formation or cleavage of covalent bonds.

Aggregation and precipitation
Protein aggregation is arguably the most common and troubling manifestation of protein instability, encountered in almost all stages of protein drug development, during refolding, purification, sterilization, shipping and storage processes (24).
The aggregation of protein molecules into non-native assemblies in vivo can have profound pathological implications, as in the aggregation of β-amyloid proteins in Alzheimer's disease and the aggregation of prion protein in numerous neurodegenerative diseases (24)(25)(26).The presence of aggregates in therapeutic protein pharmaceuticals can cause adverse effects within patients, ranging from immune response to anaphylactic shock (27,28).
Protein aggregation is defined as the association of at least two denatured protein molecules.With increasing numbers of molecules the solubility of these species will decrease, eventually resulting in the precipitation of the protein.Aggregation can occur even under conditions where the proteins native conformation is favored thermodynamically compared to the unfolded state and at concentrations well below the proteins solubility limit (24,29,30).To effectively inhibit aggregation, both in vivo and in vitro, a more complete understanding of the mechanisms by which proteins aggregate is needed (31).
Mechanisms of protein aggregation are still not fully understood.One plausible mechanism is that aggregation is catalyzed by the presence of a small amount of a contaminant.That contaminant could be a damaged form of the protein product itself, host cell proteins, or even nonprotein materials such as silica particles (32).Damaged forms of a protein product can arise from chemical modification (such as oxidation or deamidation) and from conformationally damaged forms arising from thermal stress, shear, or surface-induced denaturation.A second mechanism begins with partial unfolding of the native protein during storage.Protein conformation is not rigid -the structure fluctuates around the time-averaged native structure to different extents depending on environmental con-ditions.Some partially or fully unfolded protein molecules are always present at equilibrium in all protein solutions, but most such molecules simply refold to their native structure.However, those unfolded proteins may instead aggregate with other such molecules or may be incorporated into an existing aggregate nucleus, eventually to form larger aggregates as described above.Factors such as elevated temperature, shaking (shear and air-liquid interface stress), surface adsorption, and other physical or chemical stresses may facilitate partial unfolding.A third aggregation mechanism is reversible self-association of the native protein to form oligomers.The tendency of different proteins to reversibly associate is highly variable, and the strength of that association typically varies significantly with solvent conditions such as pH and ionic strength.Such reversible oligomers often eventually become irreversible (they are a first step along a pathway to irreversible aggregation).Detection of reversible aggregates can be especially challenging.One of the reasons is that such aggregates can dissociate from dilution during the measurement process (33)(34)(35)(36).
Regardless of the mechanism of aggregation, preventing aggregation problems requires sensitive and reliable technologies for quantitative determination of aggregate content and aggregate characteristics (37).Size-exclusion chromatography (SEC) has been a workhorse for detecting and quantifying protein aggregation (2,(38)(39).Native gel electrophoresis and SDS-PAGE have also been used to observe protein aggregation.Column-free techniques such as analytical ultracentrifugation (AUC), field-flow fractionation (FFF), and dynamic light scattering (DLS) now find increasing application in aggregation analysis (36)(37)40).As it can be concluded, there are various techniques for assessing protein aggregation, which will enable detection of dimmer and aggregate formation at the very beginning.All that is important for pharmaceutical quality and biological activity of protein -based drugs.and polysorbate-20, which are usually used in the protein formulations like surfactants, have a tendency to produce peroxides, which in turn, can lead to oxidation of the methionine residues.This peroxide contamination appears to have a greater effect on protein oxidation than the presence of atmospheric oxygen in the vial headspace or the effects of product foaming in the fill lines (14).Oxidative modification can be variable (47).Peptide maps are convenient for detecting methionine oxidation, and RP-HPLC is used to separate the oxidized forms.Oxidation causes a protein to become more hydrophilic and oxidized proteins elute before the native form in RP-HPLC (48).
Oxidation occurs in a number of proteins favored by factors like temperature, pH etc.Most proteins lose biological activity when oxidized.Thereby, formulation approaches include addition of anti oxidants (sodium thiosulphate, catalase, or platinum) and adjustment of environmental conditions (pH or temperature) to prevent oxidation (49).

Deamidation
For many proteins deamidation is one of the most often observed stability problems and occurs more rapidly than any of the other degradation routes (14).Deamidation of asparginine residues (glutamine residues to lesser extent) to aspartate or isoaspartate via succinimide intermediates (positive to negative charge) is a major cause of spontaneous degradation and loss of aminoacid sequence homogeneity.Deamidation can make protein prone to proteases and denaturation.This can affect the in vivo half-life, activity, and conformation of protein, and also increase the immunogenicity of certain protein.Deamidation often results in the loss of biological activity (50,51).
The deamidation process is mainly dependent on the storage temperature, on the pH of the formulation buffer and on the sequence and conformation.Deamidation can be detected by isoelectric focussing and quantified by densitometric scanning of the gel and by RP-HPLC (50,52,53).A common means of determining deamidation is to digest the protein with tripsin and to look for new peptide fragments eluting slightly later than fragments containing asparagine in RP-HPLC, because under acidic conditions aspartic acid is slightly more hydrophobic than asparagines (50).At neutral pH, fragments containing asparagines elute after the aspartic acid deamidation products because they are less hydrophobic under these conditions (53).

Conclusions
Proteins are only marginally stable and highly susceptible to physical and chemical degradation.Physical and chemical instabilities of proteins are the major road barriers, hindering rapid commercialization of potential protein drug candidates.Thereby, the successful development of stabile protein-based pharmaceuticals is highly dependent on a thorough understanding of their physico-chemical and biological characteristics and of the mechanisms by which proteins stabilizes.
Due to the complexity and heterogeneity of protein structures, multiple analytical methods for stability testing must be employed.Stability indicating test methods must be validated to be suitable to detect potential degradation products or modified forms.Detection of degradation products or modified forms at the very beginning signalling the stability changes important for pharmaceutical quality and biological activity of protein -based pharmaceuticals.

Introduction
It has been clearly demonstrated that the renal cells are not fully differentiated at birth and that many of the differences in renal function seen between infants and adults should be attributed to immaturity (1).This renal immaturity does not seem to carry any risk in healthy full-term infants fed an appropriate diet, but can represent a major risk in situation of disease, inappropriate fluid and electrolyte losses, and exogenous pharmacologic stress (2).
Renal insufficiency and renal failure of newborns in an intensive care unit is a very common problem.It was reported that between 8 and 24 % of neonates in special care wards ex-perience renal failure (3).Acute renal failure is a recognized complication of birth asphyxia and may result in permanent renal damage in up to 40 % of survivors (4).
Taking all these facts into account it is obvious that evaluation of renal glomerular and tubular functional and structural integrity in neonates is of great importance.For this purpose in clinical practice the investigation of a battery of biochemical parameters is frequently undertaken.
Measuring of serum concentration of creatinine, as well as cistatin C is frequently used as a parameters for evaluation of glomerular function (5).A more precise assessment of the functional capacity of the kidney is made by measuring glomerular filtration rate (GFR), but this procedure is expensive, time consuming and invasive because puncture of peripheral vein is necessary to obtain several blood samples (6,7).It is generally recognized that urine, as a medium, is a source of great number of diagnostic information.Determination of concentration of total urinary proteins, as well as measuring of concentration of different urinary proteins has been used to monitor kidney and urinary tract disease for more than 150 years.The determination of total urinary protein does not differentiate between glomerular and tubular proteinuria.It has been demonstrated that different urinary proteins carry different diagnostic information (8).So, urinary concentration of high-molecular proteins, especially albumin and immunoglobulin G, are the best markers for assessment of glomerular function.Determination of urinary concentration of low-molecular weight proteins, such as α 1 -microglobulin, β 2 -microglobulin and retinol binding protein (RBP) is recommended as potential markers for evaluating function of renal proximal tubules (9).Measuring of fractionary excretion of urinary sodium is used for investigation the functional reabsorption capacity of the distal segment of the nephron.Of all urinary enzymes, the brush-border membrane enzyme leucine-aminopeptidase (LAP) and the lysosomal N-acethyl-β-D-glucosaminidase (NAG) were recommended as markers for investigation of the structural integrity of renal proximal tubules (10).
In the recent years the accent is put on separation of urinary proteins using high resolution electrophoretic techniques, such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), capillary electrophoresis and 2D-electrophoresis (11).
The results from great number of studies have shown that SDS-PAGE is a method of choice for detection of early renal lesions (12).It is also very attractive method, because of its non-invasive nature, relatively low price and accuracy.
In the present work the patterns of urinary proteins in healthy full-term infants during the first month of life were investigated by SDS-PAGE, coupled with determination of few parameters related to urinary protein excretion.
The purpose of this examination is to evaluate SDS-PAGE profiles characteristic for healthy newborn infants; this is the basis for further evaluation of SDS-PAGE in assessment of glomerular and tubular function in conditions in which renal function might be affected (such as hypoxic ischemic encephalopathy, sepsis, nephrotoxic agents etc.).

Experimental
The present investigation was conducted on 20 healthy full-term neonates with gestational ages ranging from 36 to 41 weeks (mean 38.2 ± 1.10 weeks).
Urinary samples excreted on day 3,7,14,21 and 28 after birth were used for all examinations.Collection of urine samples was made by using a collection bag placed over the infants' external genitals.All samples were taken promptly to the laboratory and centrifuged on 3000 rpm for 15 minutes.
Total urinary proteins were determined by the Meuleman`s method, using sulfosalicylic acid (13).Urinary creatinine concentration was determined by Jaffe method (14).Proteine/creatinine ratio (P/C) was calculated by dividing the total urinary protein concentration with urinary concentration of creatinine.
Separation of urinary proteins was performed by horizontal ultrathin pore gradient-dodecyl sulfate electrophoresis according to Görg et al. (15).Urinary samples were neither diluted nor concentrated; 0.9 ml urine with 0.1 ml sample buffer (1.5 M Tris/HCl, 10% SDS, pH 8.8) was incubated for 3 minutes in boiling water.SDS-polyacrylamide gradient gels (4-22 %) were prepared by standard procedure in dimensions 195 x 250 x 0.5 mm.SDS-PAGE worked at 5 o C for 2 hours on Multiphor II Unit, LKB (Brown, Sweden).Pharmacia LKB low-molecular weight calibration proteins were used for determination of molecular mass of separated protein fractions.Gels were stained with Coomassie Blue R-250 and prepared in stable preparations.

Results and discussion
Results regarding the concentration of total urinary proteins and P/C ratio in healthy full-term neonates on days 3,7,14,21 and 28 of life are presented in Table 1.
The highest values for total urinary proteins in healthy full-term neonates were detected in urinary samples excreted on days 3 and 7 after birth with upper value of 405 mg/L and lowest values in samples excreted on days 21 and 28 of life with range between 10-50 mg/L.
The highest detected value for P/C ratio was detected in urinary samples excreted on day 3 of life (480 mg/g cre- atinine).The highest mean arithmetic value for P/C ratio was detected in urine samples excreted on day 7 after birth (297 mg/g creatinine).The differences in arithmetic means for P/C ratio between day 3 and day 28 were not so expressive like for the total urinary proteins concentration.Fig. 1, 2 and 3 present typical SDS-PAGE profiles obtained from separation of urinary proteins in neonates, included in conducted investigation.For electrophoretic separation of each subject, five different urinary samples (excreted on day 3,7,14,21 and 28 of life), with laterally applicated protein markers, were used.
In 40 % of neonates (n=8) electrophoretic profiles were characterized by the presence of proteins of mixed glomerular and tubular origin, detected in all five urinary samples.The molecular weight of proteins were found to be distributed over a wide range from 10 to 160 kDa.The intensity of fractions was highest in urine sample excreted on days 3 and 7 and decreased thereafter.On day 3 the fractions with highest intensity were those with molecular weight of 67 kDa (albumin) and molecular weight of about 30 kDa.One week after birth the low molecular weight proteins predominated because there was a substantial decrease in the excretion of albumin and of high molecular weight proteins (Fig. 1).
In 20 % of neonates electrophoretic profiles were characterized with presence of two faint fractions with molecular weights of about 78 kDa and 90 kDa, and several intensive and higher proportional low molecular weight fractions (from 14 to 67 kDa).Fractions with molecular weight of about 45 kDa, 30 kDa and 14 kDa were the most prominent in urinary samples excreted on days 3 and 7 after birth.The intensity of all fractions decreased in urinary samples excreted on days 14, 21 and 28 after birth (Fig. 2).
In other 40% (n=8) of newborns in all urinary samples only the presence of low molecular weight proteins (from 10-67 kDa) was detected.The intensity of fractions was highest in urinary samples excreted on days 3 and 7 after birth and lowest in samples excreted on days 21 and 28 of life.In 7 of neonates with this type of electrophoretic profile in samples excreted 21 and 28 days after birth, the presence of only albumin fraction was detected (Fig. 3).
Physiological proteinuria in adults is well known with many studies regarding the determination of physiological excretion of different proteins as well as separation of urinary proteins with SDS-PAGE (16,17,18).There are also a great number of studies related to quantitative changes in some specific urinary proteins during the first month of life and later in early infancy and older children (19,20).There are only a few reports in literature regarding the SDS-PAGE electrophoretic profiles in healthy newborns (21)(22)(23).
In view of the lack of knowledge about physiological proteinuria in neonates, the present study was undertaken to obtain more information about the quantity and SDS-PAGE profiles in these subjects.
The results obtained in our study for total urinary concentration have shown the highest values for TP excretion detected on days 3 and 7 with range from 10-405 mg/L which decrease thereafter with range from 10-50 mg/L on days 21 and 28.There are only a few reports related to TP concentration in neonates with different methods used for TP determination.The results in our study was in close agreement with the findings from Miltenyi (22) who reported TP concentration between 95 -455 mg/L in healthy full-term newborn infants in the first month of life (7-30 days old).
In our study the determination of proteine-to creatinine ratio was included, since the results of great number of studies of adults and children have shown that there is a strong correlation between the urine P/C ratio, obtained in random urine samples, and 24-hour urinary protein excretion corrected for body-surface area (24)(25)(26).Thus with determination of P/C ratio there is no need for 24-hour urine collection.
The highest value for P/C ratio in our study was detected in urine samples excreted on day 3 after birth (480 mg/g creatinine).In urine samples excreted on day 7 a similar values for P/C was determined as in samples excreted on day 3.In urinary samples excreted on day 14, 21 and 28 the values for P/C ratio were lower with highest detected val-ues of 415 mg/g creatinine.It is obvious that the differences between values for P/C ratios between urinary samples excreted on day 3 and samples excreted on days 7,14, 21 and 28 was no so expressive like the differences noticed for otal protein concentration expressed in mg/L for the same urinary samples.In urine samples excreted on day 3 the values for total protein concentration ranged between 10-405 mg/L and decreased rapidly thereafter in urine samples excreted on day 14 (10-120 mg/L) with lowest values in samples obtained on days 21 and 28 (10-50 mg/L).These results confirmed the usefulness of urinary P/C ratio as a better measure for urinary protein concentration, since it is very obvious that dividing the total protein concentration by urinary creatinine concentration eliminates variations due to changing rates of urine output and provides a measure independent of urine concentration.
The results obtained in our study for P/C ratio was in close correlation with the results reported by other investigators.In a study of Houser (26) the upper value for P/C ratio found in 1 month old healthy infants was 500 mg/g creatinine, which value is very similar to the values in our investigated group of healthy neonates in first month of life.
The SDS-PAGE profiles obtained in our study from investigation of the physiological proteinuria in healthy fullterm neonates reflected an incompletely developed renal function.The presence of numerous bands distributed over the broad range of molecular weights observed in urine samples from neonates could be explained with transitory immaturity of the glomerular filter and of the tubular protein reabsorbing system of the newborn kidney.Apparently, the SDS-PAGE profiles demonstrated that tubular protein handling normalizes later than the glomerular filtration of proteins.
The presence of protein fraction with molecular weight of about 160 kDa, which according to molecular weight belonged to imunoglobulin G, in some urinary samples of neonates excreted on days 3 and 7 after birth (Fig. 1), could be due to the fact that maternally derived antibodies are present at birth.Likewise, in the most of neonates prominent fraction with molecular weight of about 45 kDa was detected in urinary samples excreted on days 3 and 7 after birth, which according to molecular weight belonged to α 1 -acid glycoprotein, probably due to the acute phase response at birth.
Electrophoretic SDS-PAGE profiles, obtained in our study, have shown that the intensity of albumin fraction (67 kDa) decreased during the neonatal period, which finding could be supported by the results from quantitative determination of albumin in urine samples of neonates, excreted during the first month of life, reported by Tsukahara et al (27).The results from their study have shown that urinary albumin decreased postnatally in healthy full-term neonates, while it remained almost constant in preterm neonates.In sick pre- terms who were depressed at birth and have respiratory failure, urinary albumin was elevated during the first week, indicating the presence of glomerular damage in this period.
In almost all urinary samples excreted on days 3, 7 and 14 after birth, intensive fraction with molecular weight about 30 kDa was detected, which according to molecular weight belonged to α 1 -microglobulin.This findings is in close correlation with the results from quantitative determination of α 1 -microglobulin in urine samples of neonates, reported from Tsukahara et al. (28).The results from their study have shown high levels of α 1 -microglobulin in urine samples of neonates excreted during the first two weeks of life, which declined thereafter.This finding is due to the fact that α 1 -microglobulin was found by many authors to be early marker of renal tubular dysfunction, characterized by incomplete or inadequate reabsorption of low molecular weight proteins.
On SDS-PAGE profiles, obtained in our study, in a great number of urine samples from neonates, excreted on days 3 and 7, the presence of protein fraction with molecular weight of aproximately 12 kDa, with poor resolution, was found (Fig. 1 and Fig. 3).According to molecular weight this fraction belonged to β 2 -microglobulin, which finding could be supported by results from quantitative determination of β 2microglobulin in urine samples of neonates, during the first month of life, reported by Tsukahara et al (27).The results from their study have shown peak level on day 7 for β 2 -microglobulin, both in term and preterm neonates, with trend towards higher levels of β 2 -microglobulin with decreasing gestation, showing that proximal tubular protein reabsorption decreases with increasing degrees of prematurity.In sick preterms who were depressed at birth and had respiratory failure, this parameter was elevated during the first two weeks, indicating the presence of tubular damage in this period.
Electrophoretic SDS-PAGE profiles in 16 of neonates have shown the presence of visible, but with poor resolution protein fraction, with molecular weight of about 16 kDa.This fraction was found in all urine samples excreted on days 3,7 and 14 and in some urine samples excreted on days 21 and 28 as a palide fraction.According to molecular weight this fraction belongs to hemoglobin, which is the fraction considered to be physiological, due to the shortened life of erythrocytes (22).
Karlsson and Kristoffer (21) investigated the urinary protein pattern of pooled urine samples in the first year of life.They found a tubular type proteinuria in a pooled urine sample of eight 1-4 week old newborns, while the protein pattern of the pooled urine of eight 4-6 month old infants was identical with the pattern of adults.Their study has shown that tubular proteinuria disappears gradually during the first years of life.Miltenye and his coworkers (22) fractionated the urinary samples from 20 full-term neonates (aged from 7 to 30 days) by sodium dodecyl sulphate gel discelectrophoresis (7.5 % polyacrylamide gels stained with 0.6 % Amido Black in 7 % acetic acid).The results from their study have shown that proteinuria was predominantly tubular, although in some urine samples high molecular weight protein fractions with molecular weight bigger than 100 000 kDa was detected, as well as middle molecular weight fractions (89 000 and 80 000 kDa).They reported also the presence of hemoglobinuria during the first weeks of life.
The results obtained by analysis of the molecular weights of urinary proteins in healthy newborns, separated by SDS-PAGE and stained with Coomassie Brilliant Blue G-250, reported by Thanner et al. (23) confirmed the findings of previous mentioned authors for presence of glomerulo-tubular imbalance in early life.According to the number of separated protein fractions and their relative concentration (expressed as percent of total protein concentration) they concluded that proteinuria in healthy full-term neonates is predominantly tubular and is due to ineffective proximal tubular reabsorption of low molecular weight microproteins.

Conclusion
The results in the present study, related to examination of SDS-PAGE profiles in healthy full-term neonates, confirmed the results obtained by other investigators that electrophoretic profiles are a source of great number of information about the physiological proteinuria frequently detected in urine of neonates by screening methods.Knowledge of the modifications that occur in urinary protein pattern due to age is therefore very important in order to distinguish such modifications from those caused by other factors.Investigation of SDS-PAGE profiles, coupled with determination of few parameters related to protein excretion, such as total urinary protein concentration and proteine-to-creatinine ratio might be of great usefulness for detection of early renal lesions in neonates, in clinical condititions in which renal function is affected.

Introduction
Recent developments in technology have presented viable dosage alternatives for patients who may have difficulty swallowing tablets.Traditional tablets and capsules may be inconvenient or impractical for some patients.Geriatric and pediatric patients experience difficulty in swallowing conventional tablets which leads to poor patience compliance.This problem is overcomed with use of drug delivery system known as orally disintegrating tablets.These are novel types of tablets which disintegrate/dissolve/disperse in saliva for 60 seconds or less (1,2,3).
They are also suitable for the mentally ill, the bedridden and patients who do not have easy access to water.The be-nefits in terms of patience compliance, rapid onset of action, possibility for increased bioavailability, and good stability make these tablets popular as a dosage form of choice in the current market (2).Orally disintegrating tablets are suitable formulation for all active substances which can be orally administered (3).
A pharmaceutical formulation is composed of several composition factors and process variables.These factors and variables not only affect the characteristic property of the dosage form but also make it difficult to formulate.Formulation experience with pharmaceutical preparation generally can guide a formulation expert to select those variables that most likely have an effect on those corresponding responses (4).
Traditional formulation designs were based on trial and error.It is time-consuming, unreliable, costly and often unsuccessful.The rapid development of statistical experimental designs, optimisation techniques and computer technologies provide an effective method for modelling Macedonian pharmaceutical bulletin, 51, (1,2) 15-22 (2005) ISSN 1409 -8695 UDC: 615.214.24.014 Original scientific paper complicated multivariate drug formulations.Pharmaceutical formulation thus has been brought into a new era (5).
When a target is to evaluate one or more tablet properties, the experimenter can make use of a set of tools and techniques known under experimental research methodology.In general, adopting such an experimental approach means defining the problem which one is going to cope with by determining the objectives, the possible constraints in the component proportion and the response variables under study (6).
Many statistical experimental designs have been recognized as useful techniques to optimize the process variables.Different types of designs as screening, mixture, response surface, have been used for preformulation evaluations (7,8).
Response surface Methodology (RSM) is a widely practiced approach in the development and optimization of drug delivery devices.Based on the principal of design of experiments (DoE), the methodology encompasses the use of various types of experimental designs, generation of polynomial equations, and mapping of the response over the experimental domain to determine the optimum formulation.The technique requires minimum experimentation and time, thus providing to be far more effective and cost effective than the conventional methods of formulating dosage forms.It is a collection of statistical and mathematical techniques, useful for developing, improving and optimizing processes (9,10).It also has an important application in the design, development and formulation of new products as well as in the improvement of existing product designs.The basic components of response surface methodology include experimental design, regression analysis and optimisation algorithms which are used to investigate the empirical relationship between one or more measured responses and a number of independent variables, with the ultimate goal of obtaining an optimal problem solution.Also, interaction between different factors, which can influence the target responses, may be detected (11,12,13).
The current study aims at developing and optimizing an orally disintegrating tablet of diazepam using RSM.

Experimental Materials
Diazepam was supplied from Fabbrica Italiana Sintetici -Italy, Mannitol direct compressible grade from Merck KgA-Germany, Copovidone and Crosspovidone from BASF KgA, Germany and magnesium stearate from Mosselman, Belgium.

Experimental design
Response surface experimental design was used to optimize and evaluate main effects, interaction effects, and quadratic effects of the formulation ingredients on the percent of friability, hardness, disintegration time and drug release.A 3 2 randomized full factorial design is suitable for exploring response surfaces and constructing polynomial models (10).
In this design 2 factors were evaluated, each at 3 levels, and experimental trials were performed at all 9 possible combinations.The amount of the binding agent, copovidone (X1) and the amount of superdisintegrant, crosspovidone (X2) were selected as independent variables.The percent of friability, hardness, disintegration time and drug release were selected as dependent variables.
The composition of formulations of the factorial design is shown in Table 1.

Preparation of Diazepam orally disintegrating tablets
The raw materials were passed through a No.1.2screen prior mixing.Diazepam, copovidone and crosspovidone were homogeneously blended with directly compressible mannitol as a diluent.The mixture than was blended with the fixed quantity of the lubricant magnesium stearate and compressed into flat faced tablets (6 mm diameter) using rotate tablet compression machine.

Evaluation of tablet properties
Hardness /Mechanical strength The tablet hardness was measured by hardness tester (VanKelVK200 ,USA)

Percent Friability
The friability of tablets was determined using friability tester (VanKel, USP).The method of determination was according to USP28 method.The percent friability was obtained from 20-tablet sample.

Disintegration Time
The disintegration time was measured by using according USP 28 method for disintegration of tablets (VanKel 6-Basket System,USP)with purified water at 37 o C as disintegration medium.The mean and standard deviation were calculated from six determinations.

Percent Drug Release
Dissolution studies were performed employing USP 28 paddle method (VanKel Dissolution tester, VK-700, USA), 900 ml of 0.1mol/l HCl as dissolution medium at 50 rpm.The samples were analyzed using HPLC method (USP28).

Tablet assay
The tablets were assayed for drug content according USP 28 monograph for Diazepam tablets (Varian, HPLC system UV/vis variable wavelength detector, USA).

Statistical analysis
The data obtained from the evaluation of the tablet characteristics were analyzed using Statgraphics ® plus, Windows software program (Version 5.1, USA).

Tablet evaluation
All nine formulations of the experimental response surface design were prepared as different combinations of independent variables, copovidone (0.5-5 %) and crosspovidone (1-5 %) and the following parameters were analyzed: hardness, tensile strength, friability, disintegration and dissolution of the tablets.
The results of analysis of dependent variables-responses for each formulation are presented in Table 2.
The expressed results are mean values, for the hardness and friability determination of 20 tablets, disintegration of 12 and dissolution of 6 tablets.
During the preparation of the tablets, lower mechanical strength and increased friability for the series 1, 3, 5 under the applied pressure during tabletting were gained (RS design, quantity of the binding agent copovidone 0.5 %).Adequately, the friability of these formulations was not within the required limit, as described in USP28.
The hardness and friability were improved with increase of the concentration of the dry binder in the formulations.Complete release of diazepam from the prepared formulations was noticed after 20 and/or 30 min, showing differences in drug release rates due to different composition among series within the first 10 and 20 min.Disintegration of the tablets showing appropriate mechanical strength was fastest for series 9 (combination with 2.75 % of copovidone and 5 % of crosspovidone).However, the results given in Table 2 express that percent of drug release and the time of disintegration are not parameters that could be easily evaluated (followed) and are complex functions which mathematical expression and dependence (effect) of independent variable will be obtained from the experimental design.

Hardness of tablets
The relationship between the hardness of the tablets and the independent variables is presented in Fig. 1.
The standardized pareto chart at Fig. 1. depicts the main effect of the independent variable on the tablet hardness.The length of each bar in the graph indicates the effect of these factors and the level of their responses.From Figure 1 it can be interfered that the factor X1 has a main effect on the response Y1.Factors X2, X1X2, X2X2 did not show significant effects on the Y1 response (the hardness of the tablets).With excluding of the factors which do not have significant effect on the response, the mathematical relationship in the form of polynomial equation for the measured response Y1 would be: Y1=0.75+1*X1 (Eq. 1) Results presented in Fig. 1 and Eq. 1 show that the most significant effect on the hardness of the tablets has the binding agent copovidone, without the significant influence of the interaction between the factors i.e. between the binding and disintegrating agent.Also, Fig. 2 illustrates that the quantity of the binding agent is a factor which shows significant and linear effect on the hardness of the tablets (when applied constant punch pressure).

Friability of the tablets
The mathematical relationship in the form of polynomial equation for the measured response, Y3 is listed below: Y3=1.37834-0.750494*X1+0.0280278*X2+0.0979753*X1^2-0.00688889*X1X2(Eq.2) The polynomial equation represents the quantitative effect of process variables (X1 and X2) and their interaction on the response Y3.The values of coefficients X1 and X2 are related to the effect of these variables on the response Y3.A positive value represents an effect that favor the optimisation, while a negative value indicates an antagonistic effect.
The main effect on the independent variables on the dependent variables was further investigated using a pareto chart (Fig. 3) and interaction plot (Fig. 4).Also, the relationship between the dependent and independent variables  Biljana Nestorovska-Gjosevska, Marija Glavas-Dodov, Katerina Goracinova was further elucidated using response surface plots (Fig. 5).Fig. 3 depicts the main effect of the independent variable on the friability of the formulations.From Figs. 3 and 4 it can be interfered that the factors: X1, X1X1 (quadratic effect of copovidone), X1X2 (interaction effect of copovodone and crosspovidone) and X2 have a significant effect on the response Y3.
Fig. 5 further explains the effect of X1 and X2 ratios on the friability of the tablets.From response surface plots it is clear that the ratio of copovidone and crosspovidone has a major effect on determining friability of formulations.Figure 5 shows that at lower concentrations of copovidone the friability increases.The main effect on the disintegration time depend on copovidone and crosspovidone quantity in the formulation, interaction between the binding and disintegrating agent and quadratic effect of copovidone.Decreasing the percentage of copovidone and increasing the percentage of crosspovidone decreases the time of disintegration of the tablets (influence of the copovidone/crosspovidone interaction on the disintegration time is shown on Fig. 7).Adequately, at level of 1 % of incorporated disintegrating agent, increasing quantity of copovidone will significantly increase the value of the disintegration time for the prepared formulations (Fig. 8 shows response surface plots showing the effect copovidone and crosspovidone on disintegration).

Drug dissolution
Dissolution of the active ingredient in orally disintegrating tablets starts in the mouth after the fast disintegration where the pH is approx.6.8 (6.4-7.2).After the swallowing of the granules or the primary particles the drug dissolution continues at pH approximately 1.5 and the absorption of the released drug depends on the basic dissolution of the active ingredient and its permeability through the membranes.Diazepam is classified in the second group of biopharmaceutical classification system i.e. is practically insoluble substance at pH 6.8 or at basic pH and showing good permeability through the biological membranes.Except better patient compliance, fast dispersible/disintegration tablet means avoiding the factors of the disintegration process on the efficacy of the tablet formulation.As diazepam dissolution rate decreases at basic pH, its immediate availability in primary particles for dissolution after reaching the gaster and pH 1.5 might contribute to its better availability for absorption.
The Response Surface design shows that drug release in acidic medium is influenced only by the quantity of disintegrating agent, which is represented in Fig. 9 and Fig. 10 where factor X2 shows the main significant effect.

Conclusion
Optimization of Diazepam ODT using response surface full factorial design was performed.
The quantity and the ratio of independent variables showed significant effects on the tablet characteristics.
The most significant effect on the hardness of the tablets shows the binding agent copovidone.Increasing the quantity of copovidone increases the hardness of the tablets.
The most significant effect on the friability of the tablets has copovidone, although effect on the friability due to the interaction between copovidone and crosspovidone is also present.With increasing the content of copovidone the percent of friability of the tablets decreases.
The quantity of the copovidone and crosspovidone presents the main effect on the tablet disintegration.Interaction among copovidone and crosspovidone shows significant effect on the disintegration process, also.Increasing the quantity of crosspovidone and decreasing the quantity of copovidone decreases the time of disintegration of the tablets.Faster disintegration of diazepam tablet means that complete drug release will be achieved in medium where the active substance has a good solubility i.e. the influence of the disintegration time of the pharmaceutical dosage form on the dissolution time and of the active component is completely avoided and the dissolution rate and time will depend only on the characteristics of the active ingredient, which if needed might be modified as well.Based on the quantitative effect of the polynomial equations generated by RSM, the optimum formulation from RS design could be formulation with adequate hardness, friability and disintegration time less than 20 sec.Such formulation/s contains copovidone 2.75 % and crosspovidone in a quantity of 3-5 %.

Introduction
Hydrochlorothiazide is a thiazide diuretic drug (Fig. 1), which is used in treatment of hypertension, either alone or together with other agents, it is effective in treatment of congestive heart failure and hepatic disorders.In medical practice, it is mostly used in a conventional tablet form in dose of 25 to 250 mg (1).
Several investigations have been performed for determination of hydrochlorothiazide in urine by high-performance liquid chromatography (HPLC) with UV detection and electrochemical detection.Zendelovska

Abstract
A simple, rapid and precise HPLC method has been developed for the assay of hydrochlorothiazide in urine.The clean-up of the urine samples was carried out by solid-phase extraction using HLB cartridges.Extraction recovery was 94.00-100.28%.HPLC separation was performed with isocratic elution on Hypersil BDS C18 column (100 x 4.0 mm I.D., 3 µm particle size) protected with appropriate guard column.The mobile phase was 18 % acetonitrile and 0.025 mol/L solution of KH 2 PO 4 , pH 4 at flow rate of 0.3 mL/min.Detection of the substances was performed at 220 nm.The calibration curves were linear in the range of 2-50 µg/mL.The developed method is validated by checking its accuracy, precision and stability.The detection limit is 2 µg/mL hydrochlorothiazide.The method is proved to be convenient for routine analysis of hydrochlorothiazide in urine.mination of cephalosporin antibiotics in biological fluids and pharmaceuticals using hydrochlorothiazide as an internal standard.The confirmation technique most widely used is GC/MS, which requires methylation of the aminosulfonyl group followed by GC/MS measurement (10).

Keywords
The purpose of this study was to develop and validate a liquid chromatographic method with UV detection suitable for the determination of hydrochlorothiazide in urine.A new solid-phase extraction method was developed using HLB cartridges.This is a convenient method for separation and concentration of the analyzed drug and elimination of urine interferences, because of its efficiency and rapid sample preparation.The method was validated by evaluating the recovery, selectivity, linearity, precision and accuracy.Finally, the method was used for determination of hydrochlorothiazide in urine samples from patients.

Materials and reagents
Hydrochlorothiazide was kindly provided from Changzhou Pharmaceutical Factory (China).Acetonitrile, methanol, phosphoric acid, and potassium dihydrogen phosphate were from Merck (Darmstadt, Germany).All solvents used were of HPLC grade.HPLC column was purchased from Hewlett Packard and cartridges for solid-phase extraction were purchased from Waters.

Preparation of standards
Stock solution of hydrochlorothiazide was prepared at concentration of 2 mg/mL by dissolving appropriate amounts of hydrochlorothiazide in methanol.The working solutions were prepared by diluting appropriate portions of this solution with redistilled water and stored at 4°C.

Instrumentation and HPLC analysis
Analysis of hydrochlorothiazide was carried out with a Varian HPLC system equipped with a ternary pump Model 9012 and UV Diode Array Detector Model 9065.Satisfactory results (peak shape and run time) were obtained with Hypersil BDS C18 column (100 x 4.0 mm I.D., 3 µm particle size) protected with appropriate guard column.A mobile phase consisting of solution of KH 2 PO 4 with concentration 0.025 mol/L (pH 4) and 18 % acetonitrile was found to give the best results.Isocratic elution was performed with flow rate of 0.3 mL/min at ambient temperature.Elution was monitored in the whole UV region and the best detection was achieved on wavelength of 220 nm.
Solid-phase extraction was performed with the device (SPE) Visiprep™ from Supelco.

Sample preparation
Urine samples were collected from healthy volunteers and stored at -20°C.After thawing, samples were spiked daily with working solutions of hydrochlorothiazide.Several cartridges with different stationary phases for SPE were tested: LiChrolut RP-18 (Merck), LC-18 (Supelco) and Oasis HLB (Waters).Best results for isolation of hydrochlorothiazide were obtained when SPE was performed with Oasis HLB cartridges.
Before extraction, 1 mL of urine sample was buffered with 1 mL of 0.1 mol/L KH 2 PO 4 solution, pH 9. The cartridges were conditioned with 1 mL methanol and 1 mL redistilled water.The buffered urine was introduced into the cartridge.1 mL of redistilled water was used to rinse the cartridge and remove the interfering components.Elution was performed with 1 mL methanol.The eluate was evaporated to dryness under air for about 15 min at 40-45°C.Reconstitution was performed with 200 µL mobile phase and 20 µL sample was injected into the HPLC system.

Calibration standards
Linear regression analysis was performed.Typical calibration curves were constructed with five blank urine samples spiked with appropriate amounts of the standard solution of hydrochlorothiazide.The calibration range was 2-50 µg/mL of hydrochlorothiazide in urine.Three quality control standards containing 7, 15 and 30 µg/mL hydrochlorothiazide in urine were used for precision and accuracy measurements.

Method development
In order to develop a convenient, simple and rapid HPLC method for quantitative determination of hydrochlorothiazide in urine, several HPLC method variables with respect to their effect on the place and shape on the peak of hydrochlorothiazide, were investigated.The optimization of the HPLC conditions was performed for hydrochlorothiazide and cefotaxime, which was tested as an internal standard.Symmetrical shape of the peaks and relatively short time for analysis were obtained when separation was performed on Hypersil BDS C18 column (100 x 4.0 mm I.D., 3 µm particle size) protected with appropriate guard column with flow rate of 0.3 mL/min flow rate.The elution was monitored in the whole UV region and the wavelength of 220 nm exhibited the best detection of hydrochlorothiazide due to an absorption maximum at this wavelength.
Series of mobile phases with variable content of the organic modifier acetonitrile in range from 15 to 30 % were prepared in order to investigate its effect on hydrochloro-thiazide and cefotaxime retention.Satisfactory results (peak shape, short retention time) were obtained when the volume fraction of acetonitrile in the mobile phase was 18 %.
Additionally, the effect of the buffer concentration on analyte retention was investigated in the concentration range from 0.0125 to 0.0500 mol/L.Best results were achieved with solution of KH 2 PO 4 with concentration of 0.025 mol/L.The subsequent increasing of the buffer concentration caused increase in analysis time.Therefore, all following investigations were performed with a mobile phase, containing KH 2 PO 4 with concentration of 0.025 mol/L and 18 % acetonitrile.
The effect of the pH value on the hydrochlorothiazide and cefotaxime retention was also checked by elution with mobile phases with pH in the range from 2.2 to 4.5.A mobile phase with pH 4 provided a short analysis time and good peak shape.
Finally, the effect of temperature on retention of the analyzed component was checked in the range from 20 to 50°C.The obtained results showed that temperature does not affect the peak shape and analysis time significantly, so ambient temperature was chosen for all investigations.
From these data it was established that the best results (satisfactory retention, acceptable runtime less than 6.5 min.)were obtained using mobile phase consisting of 18 % acetonitrile in solution of KH 2 PO 4 with concentration of 0.025 mol/L and pH 4. The retention time of hydrochlorothiazide is 5.8 min and 4.0 min for cefotaxime.Typical chromatogram obtained from standard solution of hydrochlorothiazide and cefotaxime produced by the developed HPLC method is shown in Fig. 2.
Calibration diagram was constructed for establishing the dependence of the peak area of hydrochlorothiazide and concentration.The obtained linear dependence of peak area of hydrochlorothiazide and mass concentration of hydrochlorothiazide in pure solutions, with correlation coefficient (0.9999) is the following: As previously said, several cartridges for solid-phase extraction (LiChrolut RP-18 (Merck), LC-18 (Supelco) i Oasis HLB (Waters)) were tested in order to obtain satisfactory values for recovery of hydrochlorothiazide from urine samples.Results from this investigation show satisfactory values for recovery of hydrochlorothiazide when urine samples were extracted on Oasis HLB (Waters) cartridges.The values obtained for recovery of hydrochlorothiazide ranged from 94.00 to 100.28 %.The chromatographic peak is sharp, with relatively symmetric shape with small tailing, well separated form the matrix components present in the urine (Fig. 3).In this phase, cefotaxime was excluded as a possible internal standard because of its poor separation from the endogenous compounds from urine and the method was then validated using HLB cartriges for sample preparation and the optimized HPLC conditions for measurement using calibration with an external standard of hydrochlorothiazide.
Separation of interferential components from matrix was not achieved with application of RP-18 cartridges for solid-phase extraction of hydrochlorothiazide and a loss of analyzed component during the washing step was noticed.LC-18 cartridges were not convenient for separation of the analyzed component from matrix components present in urine.Endogenous components from urine were not eliminated and the chromatographic peak of hydrochlorothiazide was overlapped with chromatographic peaks of the interferential endogenous components.Intercept =-46177.9R 2 = 0.99977

Accuracy and precision
Intra-and inter-day precision and accuracy were determined by measuring urine quality control samples at low (7 µg/mL), middle (15 µg/mL) and high (30 µg/mL) concentration levels of hydrochlorothiazide.An indication of accuracy was based on calculation of the relative error of the mean observed concentration compared with the nominal concentration.Precision was expressed as relative standard deviation (RSD).Obtained results are presented in Table 1.
Relative errors ranged from 0.26 to 9.28 %, and relative standard deviation from 1.01 to 5.16 %.The obtained results indicated good precision and accuracy of the developed method.

Limit of quantification (LOQ)
The limit of quantification is defined as the lowest concentration of hydrochlorothiazide which can be measured with an error less than 20 %.The LOQ was determinated using the lowest calibration standard in five different analytical days.The RSD value of 7.57 % and a relative error less than 10 % confirmed that the hydrochlorothiazide concentration of 2 µg/mL in urine can be accepted as LOQ.

Stability of hydrochlorothiazide in urine samples
The stability of hydrochlorothiazide in urine samples was investigated with spiked samples at two different concentration levels prepared in duplicate.Spiked samples were analyzed after different storage conditions: immediately, after staying for 2 and 24 hours at room temperature, after one and two freeze/thaw cycles and after 1 month stored at -20°C.The obtained results form these investigations are shown in Table 2.
The obtained results show that relative errors are between 0.28 to 5.59 %, which means that, hydrochlorothiazide added in urine samples is stable under different storage conditions.

Ruggedness of the method
Ruggedness of the method was checked by employing the proposed HPLC method with slightly changed pH value of the mobile phase and by using slightly changed detection wavelength.The relative error was calculated by comparing the results obtained with the optimized conditions and the ones obtained with the slightly changed parameters (pH value of the mobile phase from 3.7 to 4.3, detection wavelength 215 and 224 nm).For this test, a standard solution with medium concentration of hydrochlorothiazide (7 µg/mL) was directly injected into the HPLC system.Results are present in Table 3.
The relative errors between 0.08 and 4.77 % indicating ruggedness of the developed HPLC method when using  The developed HPLC method after solid-phase extraction of hydrochlorothiazide from urine was used for analysis of patient urine samples after oral administration of the analyzed drug.Typical chromatograms obtained from urine samples prepared according to the developed procedure for sample preparation are shown in Fig. 4.

Conclusion
The HPLC method including solid-phase extraction for sample preparation is simple, rapid and convenient for determination of hydrochlorothiazide in urine samples.HPLC conditions are optimized as well as the sample preparation procedure.Different SPE cartridges for separation and concentration of hydrochlorothiazide were tested and best results are obtained with HLB cartridges.SPE conditions with HLB cartriges are optimized for separation of hydrochlorothiazide and efficient clean up of the complex biological matrix and high recovery of the investigated drug is achieved.The validation data demonstrate good precision and accuracy, which proves the reliability of the proposed method.Finally, this method is suitable for identification and quantification of hydrochlorothiazide in urine and it can be used in pharmacological studies.

Introduction
Helichrysum species (Asteraceae) have been well known plants in folk medicine for hundreds of years.One of them, Helichrysum plicatum DC. is widely distributed throughout the whole territory of Balkan Peninsula.Water extract (infuse) of this plant have been used for treatment of gastric and hepatic disorders, usually in combination with other plants with similar effects (1).Phytochemical screening (HPLC) of the Helichrysum plicatum from Macedonia proved the presence of apigenin and naringenin as free aglycones and glycosides of apigenin, naringenin, quercetin and kaempferol in the flowers as well as quercetin and luteolin glycosides and free luteolin in stems and leaves (2).Its antioxidant and scavenging properties have not been studied previously.Among phytochemicals, flavonoids deserve a special mention due to their free radical scavenging activities and in vivo biolog-ical activities that are being investigated by many researchers (3,4,5,6,7).The goal of research on antioxidative characteristics of plant extracts is to discover a potential replacement for synthetic antioxidants (BHT, BHA), which cause unwanted processes after prolonged used.
The evaluation of plant extracts antioxidant capacity is not easy task, as many methods can be used to determine this activity, and substrates, conditions, analytical methods, and concentrations can affect the estimated activity (8).This paper reports a study in which antioxidative activity of methanolic, ethyl acetate, and after hydrolysis extracts are tested.This effect was followed by three complementary in vitro methods: evaluation the free radical scavenging capacity (DPPH method) (9), inhibition of hydroxyl radicals production (10) and protection of β-carotene-linoleic acid system (11).

Plant material
The aerial parts of Helichrysum plicatum DC. were collected on the Golac Mountain, Eastern Macedonia, in the flowering period of the plant, during the summer of 2001.Voucher specimens were deposited at the Herbarium of the Institute of Pharmacognosy, Faculty of Pharmacy, Skopje, Macedonia.

Preparation of plant extracts
Dried powdered plant material (flowers, stems and leaves separately) was cut into small pieces and extracted in the ratio 1:10 (w/v) by three procedures: a. with methanol (M-extract); b. with ethanol-water (7:3), then evaporated until water remains and extracted with ethyl acetate (EA-extract); c. hydrolysis with HCl, aglycones extracted with ethyl acetate (H-extract).All extracts were dried over anhydrous sodium sulfate, filtered and concentrated under vacuum up to concentration of 1 g per 1 mL of extract.Mextract was further diluted with methanol; EA and H extracts with ethyl acetate, in order to obtained 0.01 g mL -1 solutions used in the experiments.

Free radical scavenging activity in 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) assay
The antioxidant activity using the DPPH assay was assessed by the method of Brand-Williams et al. (9).A test sample solution (200 μL) was added to 4 mL of 100 mmol L -1 ethanolic DPPH.After vortexing, the mixture was incubated for 10 minutes at room temperature and the absorbance at 517 nm was measured.The difference in absorbance between a test sample and a control (ethanol) was considered as activity.The activity was shown as IC 50 value (50% of inhibitory concentration in mg mL -1 ).Luteolin, quercetin, BHA, BHT and sylimarin (100 μg mL -1 in ethanol) were used as reference substances.All values are shown as the mean of three measurements.

Evaluation of the hydroxyl radical scavenging activity
Hydroxyl radical scavenging activity was carried out by measuring the competition between deoxyribose and the extracts for hydroxyl radicals generated from the Fe 3+ /ascorbate/EDTA/H 2 O 2 system.Attack of the hydroxyl radical on deoxyribose led to TBARS (thiobarbituric acid-reactive substances) formation (10).The extracts were added to the reaction mixture containing 2.8 mmol L -1 deoxyribose, 100 μmol L -1 FeCl 3 , 104 μmol L -1 EDTA, 100 μmol L -1 ascorbic acid, 1 mmol L -1 H 2 O 2 and 230 mmol L -1 phosphate buffer (pH 7.4), making a final volume of 1.0 mL.One milliliter of thiobarbi-turic acid TBA (1%) and 1.0 mL trichloroacetic acid (TCA 2.8%) were added to test the tube and incubated at 100 o C for 20 min.After cooling, absorbance was measured at 532 nm against a blank containing deoxyribose and buffer.Reactions were carried out in triplicate.In the series of control experiments, reference substances: luteolin, quercetin, BHA, BHT and sylimarin (100 μg mL -1 in phosphate buffer-pH 7.4) were used instead of the extract solution.The reaction mixture was incubated at 37 o C for 1 h.

Evaluation of antioxidant activity
The antioxidant activity of the extracts was evaluated using a β-carotene/linoleate model system (11).A solution of β-carotene was prepared by dissolving 2.0 mg of β-carotene in 10 mL of chloroform.One milliliter of this solution was then pipette into a round-bottom flask.After chloroform was rotary evaporated at 40 o C under vacuum, 20 mg of purified linoleic acid, 200 mg of Tween 40 emulsifier and 50 mL of distilled water were added to the flask with vigorous shaking.Aliquots (5 mL) of this emulsion were transferred into a series of tubes containing 2 mg of each extract or 2 mg of BHA (butylated hydroxyanisole) for comparison.An aliquot (5 mL) of emulsion without any further additions was used as control.As soon as the emulsion was added to each tube, the zero time absorbance was read at 470 nm.Subsequent absorbance readings were recorded at 10-min intervals by keeping the sample in a water bath at 50 o C until the visual color of β-carotene in the control sample had disappeared (about 120 min).

Results and discussion
Interest in the search for new natural antioxidants has grown dramatically over the past years because reactive oxygen species production and oxidative stress have been shown to be linked to ageing related illnesses (12).Also the restrictions to use of synthetic antioxidants, such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), due to their toxicity (13) has been an important incentive for such research.Numerous plants have been examined for antioxidant activity.According to this, antioxidant properties of Origanum spp.(aerial parts) have been studied relatively well over the past years (14,15,16).Also, Urtica sp., has been shown to have antioxidant activity (17).On the other hand, Helichrysum species have not been investigated systematically for their potential health-benefiting properties.

Free radical scavenging activity
This method is based on the reduction of DPPH, a stable free radical.Because of the odd electron of DPPH, it gives a strong absorption maximum at 517 nm by visible spectroscopy (purple color).As the odd electron of the radical becomes paired off in the presence of a hydrogen donor, that is, a free radical scavenging antioxidant, the absorption strength is decreased, and the resulting discoloration is stehiometric with respect to the number of electrons captures (18).This reaction has been widely used to test the ability of compounds to act as free-radical scavengers of hydrogen donors and to evaluate the antioxidant activity of food and plant extracts (19)(20)(21).
Free radical scavenging activity (FRSA) of Helichrysum extracts was determined by comparing with activities of substances such as luteolin, quercetin, BHA, BHT and sylimarin (reference substances), which possess some antioxidant potential.In Table 1.FRSA values obtained for plant extracts together with reference substances in DPPH assay are shown.Various extracts showed different activity in this assay.Methanol extract derived from stems and leaves showed the highest inhibitory activity with IC 50 of 6 mg mL -1 .The lowest DPPH scavenging activity was shown by ethyl acetate extract derived from flowers (IC 50 11 mg mL -1 ).The other Helichrysum extracts demonstrated similar DPPH scavenging activity (8-9 mg mL -1 ).When compared to the reference substances, the Helichrysum extracts were found to be less efficient in radical scavenging.Luteolin, quercetin, BHA, and sylimarin interacted intensively with DPPH (IC 50 : 0.09, 0.06, 0.152 and 1.96 mg mL -1 , respectively) while BHT possessed moderate scavenging properties (3.52 mg mL -1 ).The scavenging effects can probably be attributed to the flavonoids present in the examined extracts, but could also be the result of the activity of other secondary metabolites, such as volatile oils, carotenoids, and vitamins, that in the case may contributed to the antioxidant capacity (16,17).

Hydroxyl radical scavenging activity
The Fenton reaction describes the oxidation of H 2 O 2 by Fe 2+ to OH • and Fe 3+ .In the model employed in this experiment, the production of OH • induced oxidation of the deoxyribose, which in turn reacted with TBA to produce a TBA reactive chromofore that was detectable at 535 nm, thus enabling assessment of antioxidant activity of plant extracts (10).
Fig. 1 presents the results of the effects of examined Helichrysum extracts, reference substances (luteolin, quercetin, BHA, BHT and sylimarin) as well as control solution on OH • radical production.The results show that all extracts of Helichrysum plicatum inhibited the production of OH • radicals.The strongest inhibitory activity was exhibited by the H-extract derived from stems and leaves (58%).When compared to the reference substances, luteolin, quercetin, BHA, BHT and sylimarin (48%, 42%, 52%, 47% and 43%, respectively), the extracts had slightly lower activity, except of H-extract derived from stems and leaves with greater activity than BHT.
The findings demonstrate hydroxyl radical scavenging potential of Helichrysum extracts against Fenton reaction induced OH • generation, showing similar performance with luteolin, quercetin, BHA, BHT and sylimarin.This activity is mainly due to the redox properties of flavonoids, which  allow them to act as reducing agents, hydrogen donors, and singlet oxygen quenchers.They may also have a metal chelating potential (22).The slight quantitative differences in the amounts of flavonoids (2) might also explain the minor differences between the activities of the extracts.

Antioxidant activity
For the screening of antioxidant potential of Helichrysum extracts, β-carotene/linoleate model system has been applied (11).This test involves a reaction between a potential antioxidant, β-carotene and linoleic acid.β-Carotene undergoes rapid discoloration in the absence of an antioxidant.The presence of antioxidant compounds can hinter the extent of β-carotene destruction by neutralizing the linoleate free radical and any other free radicals formed within the system.
The antioxidant activity of each extract of Helichrysum plicatum, BHA and control is presented in Fig. 2. According to the preventive activity against bleaching of β-carotene, M-extract (46%) derived from flowers and H-extract (44%) from stems and leaves are the most promising Helichrysum extracts.M-extract (22%) and EA-extract (29%) derived from stems and leaves showed short and weak inhibition of bleaching, close to that of flowers EAextract (24%).H-extract (15%) from flowers was found to be less efficient in this model system.The inhibitory effect on β-carotene bleaching of the all Helichrysum extracts was lower than that of BHA.
The antioxidant activity of Helichrysum plicatum extracts has been attributed to various mechanisms, among which is neutralizing β-carotene destruction.Also, we can suggest that there is always no linear correlation between antioxidant activity and the content of flavonoids (2).This indicates that the concentration of flavonoids is not the only factor related to the antioxidant activity.Possible synergism of flavonoids with other components present in the extracts may be responsible for this observation.

Conclusion
Results of this study suggested that Helichrysum plicatum DC. could be regarded as a good source for natural antioxidant.Its extracts exhibit potent free radical scavenging, hydroxyl radical scavenging and antioxidant activity in vitro.The information from this study can explain the traditional use and the further development of these extracts into new pharmaceuticals.
The data reported here can be consider as the first information on the antioxidant properties of Macedonian Helichrysum plicatum.Further studies are needed to evaluate the in vivo potential of these extracts in animal models.

Introduction
Cystatin C (Cys C) is a nonglycosylated basic protein (13.36 kDa) and can be found in a variety of biologic fluids (1).CysC serum concentration is not influenced by gender, inflammation, or lean tissue mass and is regarded to be mainly determined by glomerular filtration rate (GFR) (2,3).Cystatin C has been described as meeting many of the characteristics of an ideal GFR marker (e.g., endogenously produced at a constant rate, freely filtered in the glomerulus, neither reabsorbed nor secreted in the renal tubule, not extrarenally eliminated) and has been reported to be at least as accurate as the commonly used serum creatinine to detect impaired renal function in various patient groups, including renal transplant patients (4)(5)(6)(7)(8)(9)(10)(11).
In recent literature, cystatin C has been advocated as a new and more accurate estimate of GFR (1).Cystatin C is a 13-kDa endogenous cysteine proteinase inhibitor produced by all nucleated cells at a constant rate and broken down completely in the renal tubuli (12).Cystatin C concentrations are independent of age and body weight, and there is no need for urine collection for clearance estimations.Furthermore, serum concentrations of cystatin C are not influenced by malignancy or inflammation.In contrast, the often-used serum creatinine concentration is supposedly influenced by dietary intake, renal tubular metabolism, age, and variations in muscle mass.There are also various analytical difficulties with the widely used Jaffe colorimetric assay for creatinine.A slight decrease in GFR has been found in patients with hypothyroidism, which improved significantly after treatment (2,3).We wondered whether cystatin C would also be a good marker of renal function in case of thyroid dysfunction.Thyroid hormones have metabolic effects and thus, thyroid state could influence plasma cystatin C concentrations.
Patients are at risk of acute damage of the transplanted kidney because of rejection or toxicity from immunosuppressant therapy.Earlier detection of renal damage may lead to more effective intervention.In a preliminary study, LeBricon et al. (13) first suggested that CysC was more sensitive than serum creatinine(SCr) for detecting decreases in GFR and delayed graft function in renal transplant patients.As in most studies, plasma CysC measurements correlated well with SCr and creatinine clearence (CrCl).However, in the three cases of acute renal rejection that were confirmed by biopsy, the increase in plasma CysC values was more pronounced than that observed for SCr.
After renal transplantation, plasma (or serum) creatinine is the most common marker for assessment of allograft function.In a steady-state muscular mass balance, the plasma creatinine concentration is assumed to reflect glomerular filtration rate (GFR) (14).However, plasma creatinine is far from being an ideal marker of GFR, despite its convenience and low cost (15) Plasma creatinine suffers a high degree of interindividual variability related to sex, age, body composition, and dietary factors (16).With altered renal function, the plasma creatinine concentration increases only when the GFR is reduced by >50%.Furthermore, secretion or reabsorption of creatinine by the renal tubule is highly unpredictable, thus leading to underor overestimation of GFR (15).
Numerous drugs and endogenous substances also interfere with the measurement of creatinine by the Jaffé technique or enzymatic methods, leading to falsely high or low creatinine values (17).
The control group consisted of 50 healthy persons.They were free of cardiac, liver or renal diseases or hypertension and had normal urine analysis and normal sera urea,creatinine and Cys C concentration.

Methods
Delayed graft function (DGF) was defined as a requirement for dialysis during the first 2 weeks after transplantation.All patients were on conventional dialysis.Episodes of acute rejection diagnosed by renal biopsy were treated with 5 days of intravenous methylprednisolone.This study was in accordance with the ethics standards of the Helsinki Declaration of 1975, revised in 1983.
Allograft function was evaluated on a daily basis starting on the day of surgery (day 0) and for 25 days thereafter or until hospital discharge, whichever occurred first.Blood (7 mL) was drawn by venipuncture in a Vacutainer® Tube (Becton Dickinson) before centrifugation (3500g at 20 °C for 15 min) and analyzed for creatinine and CysC.Creatinine concentration was enzymatically assayed on a INTEGRA biochemical analyzer.The sample volume was 10 µL, and the assay was performed at 37°C; total analysis time was 7 min.The interassay imprecision (CV; n = 50) was <3%.
The concentration of CysC was measured using a DAKO Immunoturbidimetric assay on a Cobas Mira biochemical analyser.The reference serum interval was 0.80 -1.25 mg/L.Briefly, the assay is performed at room temperature with a six-point calibration covering the range of 0.23-7.25 mg/L.The calibrator used is a purified Cys C from human urine (1.45 mg/L).The sample volume is 80 µL.The time for analysis is 6 min, each subsequent sample reading being available after 8 s.The interassay CV (n = 20) was <4% for both the low (1.4 mg/L) and high (4.2 mg/L) controls.

Calculations
The percentage of discordant changes in plasma cystatin C and creatinine concentrations was calculated on a day-to-day basis after transplantation.A change in opposite direction (increase/decrease) of >10% between the two markers was considered as discordant.On hospital discharge or at the end of the 25 days period, creatinine clearance was estimated from plasma creatinine using the formula of Cockcroft and Gault (14).A cutoff of 80 mL/min was selected for normal estimated creatinine clearance (25).

Statistical analysis
Data are presented as mean ± SD or as median and range when appropriate after checking for gaussian distribution.Differences between two groups were evaluated by the Wilcoxon's signed-rank test.Multiple comparisons were performed by the Friedman's repeated-measure ANOVA on ranks followed by the Dunn's test.Correlation between techniques was evaluated by linear regression and ANOVA.Results with P <0.05 were considered statistically significant.

Results and discussion
CysC and creatinine concentration were measured in 50 healthy adults (39 +/-9 years).Data were analyzed by linear regression and ANOVA (P < 0.05 considered significant).The concentration of Cystatin C significantly correlated (r = 0.625; P<0.001) with creatinine ( Fig. 1).Cystatin C serum concentration was not influenced by age (r=0.195;not significant).The calculated reference interval for creatinine was 58-109 mmol/L (males and females) and the cystatin C : 0.80 -1.25 mg/L.Transplant patients were separated into two groups: normal course (absent of complications; n= 38) (Fig. 2); DGF and acute rejection episode defined as requiring hemodialysis during the first 2 weeks after surgery (n=26).
In patients without DGF a significant decline in plasma concentration was more rapidly obtained for cystatin C that for creatinine.
Starting on day 4 post-transplantation the decrease in plasma concentration in patients without DGF was more pronounced for creatinine that for cystatin C (Fig. 3).
In patients with DGF, the reduction in plasma concentration was not significant until day 14 for creatnine and day 17 for cystatin C.
At the end of the 25 days -study period, a 50% reduction from the initial plasma creatinine concentration was observed in patients with DGF vs 35% for Cystatin C.The frequency of discordant daily changes in cystatin C and creatinine values was significantly higher in patients with DGF(40%) than in those without DGF (21%,P < 0.01).
In cases with acute rejection episode was demonstrated by persistent increases in both plasma creatinine and cystatin C concentration.Serum cystatin C gradually increase (115%) during 7 days before diagnosis (nephrotoxicity -FK506-regressed spontaneously).
At the end of the study (day 25), serum creatinine was 150 µmol/L (79-602 µmol/L) vs 2.12 mg/L (1.04-5.54mg/L) for cystatin C. Both markers significantly correlated (r = 0.812; P <0.001).Estimated creatinine clearance by the Cockcroft and Gault formula (14) in patients with stable renal function (no hemodialysis during the last week, n = 19) was 49 mL/min (15-66 mL/min), and no patient was within the reference interval (>80 mL/min).Serum creatinine was within the reference interval for three patients (80-109 µmol/L) compared with none for cystatin C. The relationship between estimated creatinine clearance and the serum concentrations of creatinine and cystatin C in patients with stable renal function (n = 22) is presented in Fig. 4 and 5.There was a significant correlation between estimated creatinine clearance and serum cystatin C (r = 0.714; P <0.001).

Discusion
Sensitive and reliable recognition of changes in GFR is of primary importance in transplant patients.A DGF is an identified and independent risk factor for graft survival (26) Acute rejection is also an established risk factor for renal graft failure, defined as a return of patient to hemodialysis (18)(19)(20).Recent studies have suggested that cystatin C might be a potential better marker of GFR than plasma creatinine such as in renal transplant patients (28)(29)(30).
The mean cystatin C plasma concentration in our group of healthy adults was similar to that reported in a group of healthy subjects (n = 50; mean age, 40 years), using an immunoturbidimetric assay (0.65 ± 0.05 mg/L) (30).Slightly higher values (mean cystatin C, 0.80 mg/L) were reported by others (27) in a group of 52 adults with normal renal function (ages, 21-79 years).These data are in agreement with an increase in plasma cystatin C values with age, especially after the age of 50 years (35).We found no the sex difference reported previously (22,23).Serum cystatin C correlated poorly with serum creatinine, as reported previously in subjects with GFR >80 mL/min (24) or GFR >70 mL • min -1 • 1.73 m -2 (23).These data suggest that different physiological factors (such as sex, dietary factors, or body composition) influence cystatin C and creatinine plasma concentrations in healthy adults.
In renal transplant patients, plasma cystatin C concentrations paralleled those of creatinine regardless of graft function (absence or presence of DGF).Consequently, serum cystatin C and creatinine significantly correlated over the postoperative study period as observed previously in adult renal transplant patients (31) and subjects suffering from chronic renal disease (13).Some differences, however, were apparent in their respective plasma kinetics.During the first 4 days post-transplantation, the cystatin C serum concentration decreased more rapidly than that of creatinine.Tanstubular leakage of the low-molecular weight (M r 100) creatinine (23) has been reported in acute renal failure, thus leading to high plasma creatinine values.Starting on day 4 post-transplantation, decrease in serum concentration became more prominent for creatinine than for cystatin C, which might be attributable to an underestimation of GFR by plasma cystatin C, an overestimation of GFR by plasma creatinine, or both.A stronger correlation between cystatin C than between creatinine and the measured GFR has been reported in adults suffering from renal diseases (18).
For patient follow-up, the ability to detect rapid changes in GFR is clinically more important than accuracy itself.With diminished GFR, a significant increase in plasma concentration of cystatin C and creatinine will depend on the rate of its accumulation in plasma, which depends on its production rate and distribution volume, but also on its biological intraindividual variation.Repeated measures obtained in healthy subjects (30) suggested that intraindividual variation might be more important for cystatin C (13.3%) than for creatinine (4.9%).If true, cystatin C would be less sensitive for the detection of acute rejection episodes for a given individual than creatinine (30).In all episodes of acute rejection and acute nephrotoxicity in our study, the plasma cystatin C concentration broadly paralleled that of creatinine.The rise in plasma cystatin C concentration was more prominent than in creatinine.
After renal transplantation, hemodialysis was required in almost 55% of our patients.We found a higher degree of discrepancy (45% of discordant results; P <0.01) between cystatin C and creatinine kinetics in patients requiring hemodialysis than in those with a normal course (19%).In addi- tion, cystatin C and creatinine weakly correlated in hemodialyzed patients (r = 0.429).The molecular weight of cystatin C is 13 300 with an Einstein-Stokes radius of 30-40 Å, which is much higher than creatinine (M r 100 and 3 Å) (15).In a large study of 112 patients on stable maintenance hemodialysis, a 30% reduction in serum cystatin C was observed after dialysis with mostly AN69 high-flux membranes (23).As expected, the elimination of cystatin C during dialysis increased with the ultrafiltration coefficient (UFC) of the membrane, an estimate of the permeability: 0% (vs 40% for creatinine) for UFC <15 mL • h -1 • m 2 • mmHg and ~ 60% (as for creatinine) for UFC >15 mL • h -1 • m 2 • mmHg (23).Thus, removal of cystatin C by hemodialysis seems highly dependent on the type of membrane selected.This is an important issue in renal transplantation because it could limit the use of plasma cystatin C as a marker of graft function in patients with DGF.On the other hand, if poorly filtered by dialysis membranes, plasma cystatin C could be used by the nephrologist to monitor appropriate duration of hemodialysis in patients with DGF.Prolonged unnecessary hemodialysis could be avoided as soon as a significant decrease in plasma cystatin C is obtained by dialysis.
In conclusion, the determination of cystatin C concentration is an alternative and more accurate marker of GFR than creatinine in adult transplantation.In some cases, a more prominent rise in serum cystatin C values allows a more rapid diagnosis of acute rejection or treatment nephrotoxicity.Further prospective studies are needed to evaluate this last issue and the potential of plasma cystatin C in the long term follow-up of graft function in renal transplantation.

Introduction
Cold pressed orange oil is widely used in beverage, perfumery industry and aromatherapy.In general, there are two types of orange oils: sweet orange oil from Citrus sinensis and bitter orange oil from Citrus aurantium.
Sweet orange oil is the most widely used citrus oil.It possesses a light sweet, fresh top note with fruity and aldehydic character (1).It is widely used in the flavour industry especially in beverages and candies.It can provide the top note for citrus flavouring as well as characteristic and most universally accepted flavour.The sweetness and in the same time the refreshing note makes them appropriate base for still and carbonated soft drinks.
Most of the orange oils on the world market are Italian and American industrial oils, but there are also oils produced in Spain, and in the South American countries.Moreover, studies on laboratory-extracted oils from the peel of fruits of special cultivars from different regions such as Algeria, Libya, Israel, Russia, China and Japan have been published (1).
Bitter orange oil possesses fresh citrus top notes, but is considered less sweet, and even bitter and dry.It has floral and aldehydic characteristics (1).The tenacity is greater than the most other citrus oils.In flavourings, this oil can be used to provide a citrus top note or act as a modifier in the citrus blend.The major application is in the citrus flavourings for beverages, especially liqueurs.It also intensifies the orange character in soft drinks.
Most of the papers pertinent to sweet orange oil composition were reviewed by Sawamura (2, 3) and Lawrence (4).A large number of papers deal with the composition of the volatile fraction of sweet orange oil and in many of them its differences in relation to the cultivars are reported with particular references to the different content of aliphatic aldehydes and linalool (5-10), and the volatile components of bitter orange oil (11,12).
The goal of this work is to present a way of comparative investigation of the cold pressed sweet and bitter orange oils found in the market and to see the difference between the oils.The results are compared with the published ones for fresh orange oils and some indications about the freshness and the origin are discussed.

Experimental
Two commercial samples of oils produced from peel of Citrus sinensis and Citrus aurantium were kindly supplied from an Italian flavour company.
Each sample was analysed by GC/MS using a HP 6890 Gas chromatograph equipped with a HP 5973 mass selective detector with a HP-5 fused silica column (30 m x 0.25mm, 0.25 μm film thickness).The column temperature was changed linearly from 40 to 180 ºC by 4 ºC/min, and after that 20ºC/min to 260ºC.0.2 μL were injected at a split ratio of 1:100.The inlet temperature was 260ºC and the transfer line temperature was 280ºC.
The MS library used was Wiley 275.Also, compound identification was checked by linear retention indices with standard series of alkanes (C 8 -C 32 ) and compared with the LRI values in the database of the Citrus Research and Education Centre at the University of Florida ( 13) and in the book of Adams (14).

Results and discussion
Sample preparation is one of the most important processes in flavour research, because the aroma compounds are substantially volatile and unstable against heat (3).GC-MS is a valuable tool in characterization of essential oils because the interpretation of the results of qualitative and quantitative analysis can enable an insight in the process of production, storage and age of the commercially produced oils.
In this work, more than fifty components were identified in the analysed sweet and bitter orange oils.The identified compounds and their linear retention indices calculated on HP-5 column are given in Table 1.For each sample, the quantitative composition (as a relative percentage of peak area) for each component is given together with the aroma descriptors for the components as found in the database of the Citrus Research and Education Centre at the University of Florida ( 13) and by Choi (15).
The GC-MS chromatograms obtained for both samples of essential oils of sweet and bitter orange oil are given in Fig. 1 and Fig. 2, respectively.
The data in Table 1 show that different quantities and ratios of the same components give different taste of the aroma.According to the data, the quantities of α-pinene, Fig. 1.Chromatogram of sweet orange peel oil camphene, myrcene and octanal are higher than the usual values for cold-pressed sweet and bitter orange oil (1).On the other hand, the quantities of limonene are lower (76.65 % in sweet orange oil and 71.2 % in bitter orange oil) than expected according to the published data for fresh sweet and bitter orange essential oils (over 90 %) (1).
The quantity of α-terpinolene which gives the citrus and pine note according to odor description database of Citrus Research and Education Centre ( 13) is almost six times greater in bitter orange oil than in sweet orange oil.The quantity of valencene in sweet orange oil is more than double compared to bitter orange oil, which is expected because this compound gives the specific sweet citrus odor for sweet orange.
Aldehydes are the most important compounds for the citrus flavour.The quantity of aldehydes in sweet orange peel oil is double compared to the aldehydes content in the bitter orange oil.Octanal, nonanal and decanal, which are very active odor compounds and have a sweet note, are in higher quantity in sweet than in bitter orange oil (2).
On the other hand, alcohols content in bitter orange oil is higher than in sweet orange oil, but linaool in both analysed commercial oils is in bigger quantity than expected from the published data (1).Also the quantity of esters is three times higher in bitter orange oil in comparison to quantity of esters in sweet orange oil, because the quantity of geranyl acetate and linalyl acetate are very high in bitter orange oil.
There are some components expected and identified in the in bitter orange oil like +-2-carene, n-octyl acetate, linalyl acetate, 2,4-decadienal, δ-elemene, germacren B, nerolidol, bergapten, which are missed in the sweet orange oil.β-pinene is also missed in sweet orange oil and it is present in bitter orange oil in enough high quantity.
It is well known that the bitter taste of bitter orange oil predominantly comes from the nonvolatile components of the peel, but also the volatile components give a significant part of the aroma of bitter orange oil (1).The component which gives a bitter odor, carvon, is in higher quantity in bitter orange than in sweet orange oil.It has been observed that orange oil deteriorates very rapidly in aqueous acidic environment and under the influence of light and oxygen.Relatively high quantity of carvon in both oils has been identified as the major degradation product (Fig. 3), and has also been reported as a constituent of various old citrus oils (11,16).From this we can include that the oils purchased from the market are not fresh.All these results indicate that the two commercially produced orange oils purchased from the flavour market are not fresh and original cold pressed essential oils, but some of the components, like limonene, are removed from the oil and some of the components, like α-pinene, sabinene are added to the oils by physical or chemical methods (1).

INSTRUCTIONS FOR AUTHORS
Macedonian Pharmaceutical Bulletin is an official publication of the Macedonian Pharmaceutical Association.The journal publishes original scientific papers, short communications, reviews and professional papers from all fields of pharmacyand corresponding scientific fields of interest for pharmacy (pharmaceutical and medicinal chemistry, immunology and imunochemistry, molecular biology, pharmaceutical analyses, drug quality control, pharmaceutical technology, pharmacoinformatics, biopharmacy, pharmacology, applied botany, pharmacognosy, toxicology, clinical pharmacy, food and nutrition, physical pharmacy, organical synthesis, social pharmacy, history of pharmacy etc.).
The Macedonian Pharmaceutical Bulletin, also, publishes and other contributions (recommendations and announcements, reports of meetings, important events and dates, book reviews, various rubrics).
Original scientific papers should contain own unpublished results of completed original scientific research.The number of pages (including tables and figures) should not exceed 15.
Short communications also should contain completed but briefly presented results of original scientific research.The number of pages should not exceed 5 (including one table and one figure).
Reviews are written at the invitation of the Editorial Board.They should be surveys of the investigations and knowledge of several authors in a given research area, the competency of the authors of the reviews being assured by their own published results.
Professional papers report on useful practical results which are not original but help the results of the original scientific research to be adopted into practical use.Professional papers might be based on the elaborating of theoretical data.The number of pages should not exceed 10 (including tables and figures).
Original scientific papers, short communications and reviews should be written in English, while professional papers and all other contributions may be submitted in Macedonian.
Acceptance for publication in the Bulletin obliges the authors not to publish the same results elsewhere.

Preparation of manuscripts:
Manuscripts should be submitted in triplicate, typed (1 ½ spaced) on A4 paper with margins of 3.0 cm on each side.It is recommended the manuscript be sent on the Editor-in-Chief address's: Prof. dr.Svetlana Kulevanova Faculty of Pharmacy Vodnjanska 17 1000 Skopje Republic of Macedonia The pages and appendices should be numbered.The papers should be written in the shortest possible way and without unnecessary repetition.
The names of substances should be in accordance with the IUPAC recommendations and rules or Chemical Abstracts practice.
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Manuscript should contain: title, abstract, key words, introduction, experimental, results and discussion, conclusion, acknowledgment (if desired) references and summary.
Papers should be preceded by a title page comprising: the title, the complete name(s) of the authors, and the author's affiliations (institution and address).When the authors affiliated from various different institutions the origin of the each author should be indicated with 1,2,3... at the end of his name.The correspodence author should be indicated with * and his e-mail address, telephone and fax number to be supplied in the bottom left part of the title page.
Each paper must begin with an Abstract which should not exceed more than 250 (original scientific and professional papers) or 100 (short communications) words.The abstract should include the aim of the research, methods and the most important results.A list of 3 to 6 key words arranged in the order according to their importance should follow the abstract.
In the introduction only the most important previous results related to the problem in hand should be briefly reviewed and the aim and the importance of the research should be stated.
The experimental section should be written as a separate section and should contain a description of the materials used and methods employed in form which makes the results reproducible, but without detailed description of already known methods.

Figure 1 :
Figure 1: Equation of the native and denatured state of a protein

Fig. 5 .Fig. 6 .Fig. 8 .Fig. 9 .Fig. 7 .
Fig. 5. Response surface plots showing the effect of independent variables, varying ratio of (X1) copovidone and (X2) crosspovidone on response Y3 (friability of the tablet) and Stafilov (2) published a review of HPLC methods for determination of diuretics in biological fluids including procedures for sample preparation.Liquid-liquid extraction with different organic solvents is mostly used for determination of hydrochlorothiazide in urine.But, in this way, separation of hydrochlorothiazide from interferential components is not achieved and recovery values are poor.Cooper and coworkers (3) suggested a liquid-liquid extraction of hydrochlorothiazide from urine and serum by ethyl acetate and back extraction with sodium hydroxide, but recovery values were 91,5 ± 2,5 %.Shiu and coworkers (4) proposed a liquid-liquid extraction of hydrochlorothiazide from urine with ethyl acetate, but again, the recovery values are ranged from 62.2 to 80.7 %.Alton and coworkers (5) have also used ethyl acetate for extraction of hydrochlorothiazide from urine.On the other hand, Domingo and coworkers (6) used micellar liquid chromatography for determination of hydrochlorothiazide in urine by direct injection of sample in the chromatographic system without any sample pretreatment.Zendelovska and coworkers(7)  developed a solid-phase extraction method for determination of hydrochlorothiazide in human plasma.Papadoyannis and coworkers(8, 9)  have performed a chromatographic deter-Macedonian pharmaceutical bulletin, 51, (1,2) 23-28 (2005) ISSN 1409 -8695 UDC: 615.254.1.074:543.544.5

Fig. 2 .
Fig. 2. Effect of Helichrysum plicatum extracts and control on oxidation of the β-carotene-linoleic acid model system.

Fig.
Fig. 3. Biosynhesis of limonene and its transformation to carvone during storage

Table 1 .
Responce surface experimental design

Table 2 .
Results of the determination of the dependent variables for each series

Table 1 .
Intra-day and inter-day accuracy and precision data for hydrochlorothiazide in urine determined with solid-phase extraction

Table 2 .
Stability of hydrochlorothiazide in urine

Table 3 .
Ruggedness of the method for hydrochlorothiazide at slightly varied pH values and detection wavelengths slightly variable pH values of the mobile phase.The results presented in Table3, show that little changes in the detection wavelength produce relative errors in the range from 1.09 to 4.87 % which confirms the ruggedness of the SPE method developed for solid-phase extraction of hydrochlorothiazide.
a IC 50 denotes the extract concentration at which the absorbance shows 50 % of control b All values are shown as the mean ± SD of more than 3 measurements Fig.1.Effects of Helichrysum plicatum extracts against luteolin, quercetin, BHA, BHT, sylimarin as standards and control on the in vitro OH • production.