New Trends in Sample Preparation in Brazil : an Overview of Bioanalytical Applications by Liquid Chromatography

Sample preparation is an integral part of the analytical process and it is by far the highest problem in bioanalysis, furthermore is considered the most environmental pollutant step of the analysis. All the talk of reducing the tedious sample preparation step by liquid chromatography coupled to tandem mass spectrometry has proved to be no more than a wild dream of fancy. On the other hand, sample clean-up procedures yielding higher analytical turnover with low limits of quantification are of utmost importance. The goal of the analysis influences the procedure used in sample preparation. In bioanalysis, it usually involves fractionation, isolation and enrichment of the target analytes from the matrix. Most of these protocols are performed off-line affecting time and the cost of the analysis. Green analytical chemistry is a growing concept in separation science. Thus, considered as a priority, new approaches to decrease risk contamination and waste have been pursued, with new technologies for direct sample injection or miniaturization of well settled approaches. Automated sample preparation has also been widespread used, and scores of reviews have covered this important topic. We shall henceforth outline the Brazilian contribution to this subject throughout the last decade. 2. Restrict Access Media (RAM)


Introduction
Sample preparation is an integral part of the analytical process and it is by far the highest problem in bioanalysis, furthermore is considered the most environmental pollutant step of the analysis.All the talk of reducing the tedious sample preparation step by liquid chromatography coupled to tandem mass spectrometry has proved to be no more than a wild dream of fancy. 1 On the other hand, sample clean-up procedures yielding higher analytical turnover with low limits of quantification are of utmost importance.
The goal of the analysis influences the procedure used in sample preparation.In bioanalysis, it usually involves fractionation, isolation and enrichment of the target analytes from the matrix.Most of these protocols are performed off-line affecting time and the cost of the analysis.
Green analytical chemistry is a growing concept in separation science. 2 Thus, considered as a priority, new approaches to decrease risk contamination and waste have been pursued, with new technologies for direct sample injection or miniaturization of well settled approaches.4][5][6][7][8] We shall henceforth outline the Brazilian contribution to this subject throughout the last decade.

Restrict Access Media (RAM)
RAM phases have been explored for direct sample injection since their commercial introduction at the end of eighties.The unique feature of these columns is that they exclude the macromolecules of the matrix while selectively retaining the small molecules. 5,8,9In 1985, Hagestam and Pinkerton introduced the internal surface reversed phase (ISRP) support by Regis Technologies, and later on Boos et al. prepared the alkyl-diol silica (ADS) phase, commercialized as LiChrospher ® ADS by Merck. 9Among the variety of RAM sorbents, proteins-based phases were one of the first type, being introduced by Yoshida and coworkers. 10Avidin and α 1 -acid glycoprotein RAM columns were commercialized later on. 9The RAM bovine serum albumin (BSA) phases were initially criticized, for they had not great stability.The work of Menezes and Felix 11 changed it all by cross-linking the protein with glutaraldehyde.The RAM-BSA columns have demonstrated high efficiency for protein depletion using only water as mobile phase. 12The RAM-BSA columns prepared in accordance with Menezes and Felix protocol 13 has no need of buffer for salting out the sample proteins. 14,15Their smooth and relatively easy preparation 14,16 with high reproducibility makes them an excellent choice for direct sample injection.The RAM-BSA columns were successfully explored for assessing, with a series of Brazilian volunteers, 17 the enantiomeric ratios of the proton pump inhibitors (PPIs) pantoprazole, 16 omeprazole 18 and lansoprazole. 19For that, RAM-BSA-C 8 columns were coupled to polysaccharide chiral columns, in a two multidimensional liquid chromatography (2D LC) configuration. Figure 1 illustrates the quality of samples analyses.In spite of the higher number of work using RAM columns for protein depletion, few works deals with chiral separation, the PPIs works were among the first. 9To explore other RAM hydrophobic phases BSA-C 18 , BSA-phenyl and BSA-CN phases were prepared and their capacity for depletion of plasma and bovine milk protein evaluated. 12,20rotein depletion was obtained with high percentage with all columns using only water as mobile phase.
The RAM-BSA-C 18 column coupled to a C 18 analytical column was used for quantification of amoxicillin in human plasma. 21To extract amoxicillin with high precision and high recovery, it was necessary ion pairing (by addition of a cationic counter-ion) in the sample, and the use of 0.01 mol L -1 phosphate buffer, pH of 7.2, as mobile phase, at protein depletion stage.
A RAM-BSA-phenyl column coupled to a C 18 analytical column was efficiently used for quantification of cefoperazone in bovine milk. 20For quantifying cephalexin, a RAM-BSA-C 8 was used in the first dimension. 22o further explore 12,20 the RAM-BSA columns retention capability of small hydrophilic molecules in samples with high protein content, 2D LC methods were employed for the simultaneously quantifying sulfamethoxazole and trimethoprim in bovine milk 23 and in whole eggs 24 samples.The on-line sample clean-up procedure was carried out also for quantification of these bacteriostatic drugs in bovine milk while employing amperometric detection at a borondoped diamond electrode. 25he efficient coupling of RAM-BSA columns to polysaccharide columns granted their use for simultaneous quantification of enantiomeric mixtures and their metabolite in human 14,26 or bovine 27 plasma.For modafinil enantiomers and its two major metabolites, the small RAM-BSA-C 8 column (10 × 4.6 mm i.d., 10 µm, 100 Å) was coupled to an amylose tris[(S)-1-phenylethylcarbamate] chiral column.The quality of the performance of both columns was maintained with over 280 plasma injections of 100 µL, each. 26eanwhile, Santos-Neto et al. 28,29 demonstrated the viability of using RAM columns in a capillary approach.For that, they explored a 25 cm fused silica capillary tubing of 50 µm i.d.coupled to a YMC ® ODS-AQ capillary analytical column (5 µm, 120 Å; YMC Europe) which is coupled to an electrospray interface tandem mass spectrometry (ESI-MS/MS).As RAM phases, they used a LiChrospher ® ADS-C 18 (25 µm, 60 Å; Merck), SPS ® C 18 (Semi-Permeable Surface, 5 µm, 100 Å; Regis Technologies), and the in-house prepared bovine serum albumin-C 18 (BSA-C 18 , 10 µm, 120 Å).Throughout these approaches, the back-flush mode was preferred for coupling the columns in the 2D LC system. 28,30,31Lower sample dilution and consumption of mobile phase are the main attractive of the capillary approach procedure. 28Furthermore, the use of ESI-MS/MS furnished limits of quantification of 1.0 ng mL -1 for five antidepressant drugs, with injection of 1 µL of sample. 30he RAM columns are usually used in the LC-LC 2D configuration, due to lower selectivity. 5,9][34][35] A RAM-BSA-C 18 (50 × 4.6 mm i.d., 10 µm, 100 Å) column was used in the single mode coupled to an ion trap mass spectrometry for measuring both carbamazepine and its active metabolite carbamazepine 10,11-epoxide, in human milk. 36Lipids and proteins are the main components of breast milk interfering in drug recovery and their detection by MS/MS.The molecular mass cut-off of the RAM-BSA-C 18 column for the human milk proteins were evaluated by the elution profile of β-casein (24,000 Da) and α-lactalbumin (14,178 Da), at concentrations of 5.0 g L -1 , examined at the same conditions used for the human milk samples.Extraction efficiency, accuracy, and precision were achieved employing 100 µL of the sample.
The use of ultra high performance liquid chromatography (UHPLC) tandem mass spectrometry for the fast analysis of small molecules within complex matrices, with lower quantification and detection limits, is already well established. 8,37The methods for sample clean-up, however, have remained a drawback for such procedures.It has been recently reported on the use of an injection/column-switching system for coupling a RAM-BSA-C 8 , (50 × 2.1 mm, 10 µm, 100 Å) to an Acquity UPLC ® BEH C 18 (50 × 2.1 mm, 1.7 µm; Waters) column. 38The method requires a 14 min analysis, and the RAM-BSA columns have once more proven to be capable of fast on-line protein depletion.
A recent review about 2D LC-MS, 8 has drawn attention to procedures such as turbulent flow injection that usually requires pretreatment, while with RAM columns this is not necessary.
In spite of published works on environmental samples, 39,40 no papers on 2D LC-MS chiral bioanalytical applications have been published by the Brazilian researchers.Considering the expertise in the field, we expect to see further applications on chiral separation.

Molecularly Imprinted Solid-Phase Extraction (MISPE)
Molecularly imprinted polymers (MIPs) are synthetic polymers with high specific molecular recognition capability.MIPs preparation is usually carried out by polymerization of a monomer around a template using a cross-linker in the presence of an initiator.][43] The use of solid-phase extraction based on MIPs has been explored for a variety of analytes within different biological matrices. 42,43In this respect, Vieira et al. 44 reported an off-line MISPE procedure for the selective extraction of trans,trans-muconic acid from urine samples followed by LC-UV analysis.Their method exhibited good sensitivity in applications within occupational and environmental toxicology.A cotinine-imprinted polymer was also developed for the off-line quantification of cotinine in saliva samples by LC with diode array detection (LC-DAD). 45The method was successfully applied for extracting cotinine from smoker's samples.Recently, Melo and Queiroz 46 developed an off-line MISPE to preconcentrate parabens from human milk samples.The MISPE⁄LC-UV was adequated to determine methyl, ethyl and propyl parabens and compared with the classical extraction methods, the miniaturized approach minimized the volumes of organic solvent and biological fluid.

At-Line Solid-Phase Extraction (SPE)
The at-line approach is usually mixed-up with the online one.The difference between them is, however, well settled.In the first case, there is the automation of the SPE process, while in the second case there is a multimodal LC-LC system.
At-line SPE 47 is an important approach for labor-intense analysis, furnishing high throughput with high precision and sensitivity.In this context, it has been used for clinical analysis and in bioequivalence studies.Carvalho et al. 48ave quantified endogenous adrenal steroids and have drawn attention to the quality of such technique as an alternative to immunoaffinity assays and gas chromatography-mass spectrometry (GC-MS) for steroids clinical profiling.Works have demonstrated that the SPE at-line with a LC-MS/MS is an important tool for bioequivalence studies, whereby thousands of samples are processed with minimum of manual operation.[49][50][51][52][53] In Brazil, this sample preparation approach has been explored also for pharmacokinetics studies, as in the quantification of gatifloxacin in rat plasma 54 and of etoricoxib in human plasma.55

Solid-Phase Microextraction (SPME)
SPME was first introduced in the early 1990s by Pawliszyn's group as a new and effective sample preparation method to solve problems commonly associated to solid phase extraction, such as: high blank values, variability among the cartridges from different manufactures, and interferences due to adsorption of analytes on the SPE cartridges. 7,56In the SPME approach, fused silica capillary are coated on the outside with an appropriate stationary phase, and the analytes are adsorbed by simple exposure to fiber.For gaseous samples, headspace is used, while direct immersion is employed for liquid samples. 4,6,7To meet this end, analytes' partition and desorption are involved. 57nitially, the SPME was developed for the analysis of organic compounds from aqueous sample matrices using GC. 56Later it was applied for preconcentration of drugs from biological fluids by GC or LC. 58While in GC analysis the analytes are thermally desorbed by a heat chamber, 56 without solvent, in LC the desorption is carried out either off-line or on-line mode in a suitable volume of selected solvent. 6,7he success of the SPME is determined by the physicochemical properties and the thickness of fiber coatings. 6The variety of commercially available coatings has contributed to the number of classes of analytes that can be successfully analyzed in different matrices.For example: polydimethylsiloxane (PDMS) for extraction of non-polar analytes; polyacrylate (PA) and polydimethylsiloxanedivinylbenzene (PDMS-DVB) for extraction of polar analytes, especially phenols and amines, respectively; carboxen-polydimethylsiloxane (CAR-PDMS) for extraction of volatile/low molar mass analytes; carbowaxdivinylbenzene (CW-DVB) for extraction of polar analytes (especially alcohols); carbowax-templated resin (CW-TPR) for extraction of polar analytes; and divinylbenzenecarboxen-polydimethylsiloxane (DVB-CAR-PDMS) for extraction of broad range of analytes.Other less frequently used coatings include carbon nanotubes, several crown ethers, MIPs, anodized metals, and ionic liquids. 4,6 Brazil, the first work using off-line SPME for LC bioanalysis was reported on the determination of lamotrigine, carbamazepine and carbamazepine 10,11-epoxide in human plasma. 59Silva et al. 60 and Cantú et al. 61 reported the use of off-line SPME-LC on the determination of antidepressants and anticonvulsants in plasma samples.Both works showed high sensitivity and reproducibility on the quantification of these drugs in human plasma with no special interface.A comparison between commercial (PA, PDMS-DVB, CW-TPR) and inhouse fibers (polyurethane, octadecylsilane, aminosilane) was carried out by Queiroz and co-workers. 61The in-house fibers gave higher quantification limits than the commercial ones; the authors stressed, however, the benefits of in-house fibers, such as, easy preparation, good mechanical strength and low cost.Regardless of all desorption inconvenient in the off-line mode, and the increase in solid residue, an advantage is that the use of multiple SPME fibers, improves the throughput analysis. 7ased on the Pawliszyn's work, 62 Lanças and co-workers 63 developed a new heated interface with lower inner volume for on-line coupling in order to minimize common problems related to the commercial SPME-LC interface.Improved sensitivity, qualitative and quantitative results were obtained for fluoxetine analysis when compared either to an interface without heating or to SPME extraction in the off-line mode.
Table 1 summarizes the SPME Brazilian methods based on the use of heated interface for drug analysis in biological matrix.
Queiroz and co-workers 68,69 evaluated the use of new phases coating, polypyrrole (PPY) and polythiophene (PTh) prepared on a stainless-steel wire by electrochemical polymerization.The PPY fiber offered higher sensitivity (in order of 10 times more) due to the matrix effect observed in the quantification of common antidepressants (citalopram, paroxetine, fluoxetine and sertraline).
1][72] These works deal with the quantification of chiral drugs and their metabolites in human urine, by off-line approach, using CW-TPR, PDMS-DVB and PA fibers.Good sensitivity, selectivity and less consume of organic solvents were achieved.

In-tube Solid-Phase Microextraction (in-tube SPME)
This technique uses capillary columns for on-line analyte extraction.In-tube SPME systems can be classified as flow through or draw/eject.In the flow through mode, the sample solution is passed continuously in one direction through an Vol. 25, No. 1, 2014   extraction capillary column, which is placed in an automatic six-port valve.In the draw/eject extraction systems, the capillary column can be installed between the injection loop and the injection needle of the LC autosampler.][75] Capillaries prepared with selective materials, such as immunosorbent, PPY polymer and RAM sorbent, have been developed to improve both extraction efficiency and selectivity.The immunoaffinity capillaries SPME devices were used for quantifying fluoxetine in serum samples 76 and interferon alpha 2a in plasma sample. 77The drug were extracted using draw/eject procedure and the separation was carried out at a C 18 column.
An in-tube SPME PPY-coated capillary was prepared in-house in order to analyze fluoxetine and norfluoxetine enantiomers in plasma samples.The capillary was used in a draw/eject system using a Chiralcel  OD-R as the analytical column. 78The developed method was employed for monitoring patients under fluoxetine therapy (Prozac  , 20 mg day -1 ).
The same research group has also prepared a RAM-BSA-C 18 in-tube-SPME capillary for the determination of interferon alpha 2a in human plasma. 79A single draw/eject cycle was used for sample extraction while the analysis was carried out at a LichroCART  RP-18 column.The developed method has adequate sensitivity and selectivity for therapeutic monitoring of interferon alpha 2a in human plasma samples.
The main drawback of in-tube SPME is the requirement of very clean samples, since the capillary column can be easily blocked.4][75] The biocompatibility of the RAM-BSA support 79 allowed the direct injection of plasma samples with no sample manipulation other than dilution, reducing, thus, the total analysis time and in accordance with the green analytical chemistry aim.
The works carried out in Brazil using on-line in-tube SPME system are summarized in Table 2.

Stir Bar Sorptive Extraction (SBSE)
The SBSE was introduced in 1999 by Baltussen et al. 83 as a solventless sample preparation method.Magnetic coated stirs bars are the main attraction of this technique. 84n SPME, the maximum volume of coated PDMS on to the fiber is of 0.5 µL (film thickness 100 µm), whereas, about 25-125 µL of PDMS are used for the stir bars in SBSE, increasing the extraction efficiency. 6,85,86ne limitation of SBSE, when compared to SPME, is that there is only one commercial available extraction sorbent, PDMS, although in different length and thickness. 84iming to increase the applicability of SBSE, in-house sorptive phases have been developed.Accordingly, Melo et al. 87 produced a dual-phase polymeric coating consisting of PDMS and PPY for antidepressants extraction (mirtazapine, citalopram, paroxetine, duloxetine, fluoxetine and sertraline) from human plasma.The PDMS-PPY coated stir bar showed high extraction efficiency (sensitivity and selectivity) toward the targets analytes.
The quantification of ivermectine in bovine plasma was carried out using a PDMS modified bars with 5% of polydimethylphenylsiloxane and 10% of polydiethyleneglycol succinate.They showed higher efficiency when compared with the commercial PDMS bars. 85n Brazil, SBSE has been used mostly for analyzing antidepressants, 88,89 anticonvulsants 90 and antituberculous drugs in biological matrices. 91

Microextraction by Packed Sorbent (MEPS)
MEPS are the miniaturization of SPE and works with reduced sorbent bed volume, and they are suitable for a large sample volume range (10-1000 µL).Since they are integrated in the syringe, they diminish the number of steps typically involved in the conventional SPE, and are easily automatized. 6Sorbent materials such as silica based (C 2 , C 8 , C 18 ), strong cation exchanger (SCX), RAM, hydrophilic materials, carbon, polystyrene-divinylbenzene copolymers, and MIPs can be used. 92,93he main advantage of the MEPS, when compared to conventional SPE, is that the packed syringe is used several times, more than 100 times for plasma or urine samples, without loss of performance.A protocol for guaranteeing the performance of MEPS cartridges is, however, necessary, which includes dilution, centrifugation, and precipitation steps. 93Chaves et al. 94 reported the use of MEPS based on C 8 /SCX for the extraction of sertraline, mirtazapine, fluoxetine, citalopram, and paroxetine in human plasma.Effects on the extraction efficiency were examined for sample volumes, pH, number of extraction cycles, and desorption conditions.Different sample pre-treatment was also evaluated, such as precipitation, centrifugation and dilution.Dilute samples (1:1, v/v) led to sensitive, selectivity, and accurate quantification of the selected drugs.Furthermore, the cartridge might be reused more than 50 times.
MEPS cartridge (C 8 /SCX), as sample clean-up procedure, was used for the first time by Salami et al. 95 for the simultaneous determination of sulfonamides in whole egg samples.Precipitation and centrifugation were, however, used as sample pre-treatment.The cartridges were reused more than 60 times with minimum loss of extraction efficiency.
A C 18 cartridge was used by Bordin et al. 96 for determining voriconazole in oral fluids and plasma.The extraction variables parameters such as, pH of the buffer used in the sample, number and flow rate of extraction cycles were examined.For the analysis, the pH was the most important factor.Extraction time was of 4 min per sample.The cartridge was used for about 40 extractions.
An important issue to be noticed is that MEPS efficiently reduces the organic solvent volume used and the amount of solid residue.Thus, it can be considered as a green chemistry approach.

Hollow Fiber Liquid-Phase Microextraction (HF-LPME)
HF-LPME developed in 1999 by Pedersen-Bjergaard and Rasmussen 97 uses a water immiscible organic solvent immobilized as a thin supported liquid membrane (SLM) in the pores of a hollow polypropylene fiber.For extraction, the lumen of the hollow fiber is filled with a small volume of an acceptor phase (usually in the range 2-30 µL), and the whole assembly is placed in the matrix sample (typically within 100 µL to 4 mL).The analytes are extracted from the aqueous sample (donor phase), through the organic SLM, and further into the acceptor phase (aqueous or organic) inside its lumen.After extraction, the acceptor solution is collected and analyzed. 98,99F-LPME can be performed in either two or threephase mode.In the first mode, the acceptor solution and the immobilized organic solvent are the same.They may be employed for compounds with high solubility in nonpolar organic solvents, and acidic/basic analytes.[100][101] Different research groups in Brazil have employed two and three-phase HF-PLME for the determination of drugs and their metabolites in biological fluids, as the HF-LPME technique allows high analyte enrichments, despite UV detection being known to provide relatively poor sensitivity (Table 3).Bonato and co-workers have used HF-PLME procedures followed by LC analysis for quantification of drugs in different biological matrices using either chiral columns (macrocyclic antibiotic and polysaccharide-based stationary phases) [102][103][104][105][106][107][108][109][110][111] or achiral columns. 112,113iqueira and co-workers 114 and Porto et al. 115 employed three-phase HF-LPME, coupled to LC-fluorescence detection, for the analysis of fluoxetine/norfluoxetine, and citalopram, paroxetine and fluoxetine in human plasma, respectively.Both methods showed excellent sample clean-up, selectivity and sensitivity.
High recovery associated with low organic solvent consumption is the main attractive of this sample clean-up procedure.Furthermore, its application in sample clean-up procedures for chiral bioanalysis with UV detection demonstrates the high capability of producing clean samples.Chiral selectors easily lose selectivity, demanding generally cleaner samples.

Final Considerations
Greener analytical procedures are characteristic to miniaturization, to at-line and on-line systems, due to reduced generation of waste.2D LC systems have proven their ability in producing on-line matrix protein depletion, and their versatility has been efficiently explored by Brazilian researches for a variety of bioanalytical applications.The use of capillary columns, in-tube SPME, and the coupling of a RAM to a UHPLC column have demonstrated that the researchers are not only interested in efficient sample enrichment, but in producing less waste.
The trends on sample preparation, as here reported, reflect the effort of achieving green parameters and getting cleaner samples with overall smaller analysis time.

Figure 1 .
Figure 1.Chromatograms of plasma samples from a volunteer collected after an oral dose of 40 mg of omeprazole.

Table 2 .
Use of in-tube SPME in biological sample

Table 3 .
Application of HF-LPME in biological sample