Amino acid reference intervals by high performance liquid chromatography in plasma sample of Brazilian children

Introduction: The high performance liquid chromatography is a technique used for quantification of amino acids in plasma. The definition of reference intervals in the population is very important for the diagnosis and monitoring of individuals with amino acid disorders. Objectives: The objectives of this study were to validate a method for amino acids quantification and define reference intervals in Brazilian children. Results: Good chromatographic separation was achieved using C18 solid-core column. The method showed satisfactory linearity, limits of detection and quantification, precision and accuracy. The reference ranges for aspartate, glutamate, asparagine, histidine, serine, glutamine, arginine, tyrosine, alanine, tryptophan, methionine, valine, phenylalanine, isoleucine and leucine were defined in a group of 60 healthy individuals aged 2 to 14 years. Conclusion: The presented technique can be applied in clinical practice. Each laboratory should preferably establish its own reference intervals. If that is not possible, it is recommended the use of the reference intervals described in this study for the diagnosis and monitoring of Brazilian children in this age group.


INTROduCTION
Inborn errors of metabolism constitutes a diverse group of individually rare disorders, that group correspond to 10% of the genetic diseases described (1) .They all occur due to enzyme defects that cause disruption of specific metabolic pathways (2) .Within this group, the amino acids disorders stand out as serious diseases, whose symptoms are caused by acute or chronic poisoning by the accumulation of amino acids and other toxic metabolites, which might damage the brain, kidneys and liver (1,3) .Their relative rarity and nonspecific presentation may contribute to the delay or the difficulty to diagnosis, which leads, sometimes, to irreparable consequences on child development (4)(5)(6) .
Thereby, quantitative analysis of amino acids in blood and urine are tests especially indicated for patients suspected of amino acid disorders and organic acidemia.These exams can also be applied in the management of patients with these diseases that make use of dietary formulas restricted in amino acids and also for therapeutic decisions during decompensation and crisis (7,8) .A decrease or mild elevation of amino acids level can only be detected in blood, and morning fasting blood specimens are preferred.High performance liquid chromatography (HPLC) with derivatization is widely used for the diagnosis of most amino acid disorders.
Amino acids levels must be cautiously interpreted, since the levels of these substances in the blood are influenced by a number of factors, such as age, physiological changes, nutritional status, illnesses, medications and toxins.Furthermore, there are no wellestablished reference intervals in our population, and data from international literature are traditionally used (9)(10)(11)(12) .The aim of this study was, therefore, to validate a technique of measurement of amino acids in plasma using HPLC with fluorometric detector and solid-core column, as well as define reference ranges for serum amino acids for the pediatric group from the population studied.

Equipment
The equipment used was the HPLC Alliance HT Waters ® 2695 (Milford, USA) equipped with quaternary pump, thermostat, automatic injector, degasser and fluorometric detector multivariable model 2475.The system was controlled by Empower 2.0 software.

Solutions
The derivatization solution was formed by OPA (0.02 g) prepared with mercaptoethanol (20 µl), ethanol (100 µl) and 0.4 M borate buffer in alkaline environment (pH 9.5 to 1 ml).Solutions of 20% formic acid, 20% methanol and 50% acetonitrile were utilized to prepare stock solutions of each amino acid (Table 1).The intermediary solutions and solutions used were prepared with reagent grade water.

Preparation of mobile phase and chromatographic conditions
The gradient mode (Table 2) was used for better separation of amino acids and two mobile phases were prepared.The mobile phase A composition was phosphate buffer, methanol and water (57/33/30% v/v) with pH 6.5 and mobile phase B composition was methanol and phosphate buffer (47/53 v/v%) with pH 8.5.The wash solution was constituted by methanol 10%.The solid-core column AccuCORE C18 (150 mm × 4.6 mm, particle size 2.6 mM) of Thermo Scientific ® (Waltham, USA) was used to achieve separation of the amino acids.The wavelength of the detector was l ex = 330 nm and l em = 418 nm.

Validation methods
After method optimization, statistical tools were applied using the software Statistical Package of the Social Sciences 12.0 ® (SPSS).
The linearity was studied in four concentration levels.The analytical curves and the coefficient of determination (r 2 ) were obtained and the method was considered linear when r 2 was greater than 0.99.

Limit of detection (LOD) and quantification (LQ)
To evaluate the LOD and LQ of the method, 10 independent replicates were prepared from the white plasma pool.The solutions were processed as described in the topic "Sample collection and processing".A signal-to-noise ratio of 3:1 was used to determine the LOD and a ratio of 10:1 was used to determine the LQ of the method.
For precision, the solutions were analyzed by chromatography on the same day of preparation (intra-assay precision) and on three consecutive days (inter-assay precision).The intra-assay and inter-assay precision were expressed by the coefficient of variation.The accuracy of the method was verified by triplicate analysis of each amino acid and the recovery percentage was calculated.

Ethical aspects
This study was approved by the Research Ethics Committee of the Universidade Federal de Minas Gerais (UFMG) under registration number 08169212.2.0000.5149and all participants and/or guardians signed the informed consent form.

Population
A cross sectional study was carried on with individuals selected in the Hospital das Clínicas of the UFMG, among children from primary and secondary care units linked to the institution.The group was formed by 60 individuals aged between 2 and 14 years, of both genders.The sampling was done considering the exclusion and inclusion criteria and the period of two months available for the study, characterizing a convenience sampling.All children in this age group who attended in central laboratory for blood collection were interviewed for the identification of any exclusion criteria.

Inclusion criteria
Healthy children who attended the Hospital for performing routine checkups were included.All subjects responded to a questionnaire on date of birth, previous diseases, use of medications, fasting time, and presence of vomiting.

Exclusion criteria
Children with history of short stature or insufficient weight gain, who presented fasting or vomiting lasting more than a day or who were in use of interfering substances during the collection period, such as ascorbic acid, aspartame, aspirin, sulfamethoxazole/trimethoprim, glucose, indomethacin, progesterone, testosterone and valproate were excluded.Patient in pre or post-transplant, with liver or kidney disease, type 1 diabetes, sickle cell anemia, leukemia and other cancers, cystic fibrosis, genetic syndromes and dysmorphisms, acquired immunodeficiency syndrome (Aids), syphilis, visceral and hematological alterations under investigation, autoimmune diseases, endocrine and hormonal disorders were also excluded (13) .Younger children, especially under one year, were not included in the group, because blood levels of amino acids present greater variation in these children compared with the age group studied (13,14) .

Sample collection and processing
The sample collection was performed after fasting of 8-12 hours.For each patient were collected 4 ml of venous blood in a Vacutainer ethylenediaminetetraacetic acid (EDTA) blood collection tube.The sample were centrifuged at 2,000 rpm for 5 minutes to separate plasma and immediately frozen at a temperature of -20ºC until the time of analysis, which was carried out in a maximum period of 15 days.Sample preparation consisted of adding 20 µl of plasma to an Eppendorf and 100 µl of the derivatizing solution previously described.The solution was homogenized with the aid of a pipette and transferred into an insert.After 1 minute, 1 μl of the solution was injected into the chromatograph.

Statistical analysis
For definition of the reference ranges, statistical analysis was performed using the software Medcalc ® .The detection of probable outliers was tested by Reed's method (15) .The reference ranges were calculated using the robust method, after logarithmic transformation when the raw data were not parametric according to Kolmogorov-Smirnov test (16) .For data that did not show parametric distribution, even after transformation, nonparametric methodology was used to calculate the lower limit, which corresponds to 2.5 percentile.The upper limit was calculated by the robust method, according to the method described by Horn and Pesce (2005) (17) .Confidence intervals of 90% for the lower and upper limits of each interval were generated, except when the non-parametric method was applied, because n < 120 (16) .The Student's t test and Wilcoxon test were applied to comparison with the literature.

Method validation
After optimization, the run time was 90 minutes and the gradient established allowed a good chromatographic separation of the validated amino acids (Figure 1).The amino acids glycine and threonine showed analytical signal amongst amino acids arginine and tyrosine, but did not provide good chromatographic separation and were not included in this validation.Linear responses were found with r 2 > 0.99 for all amino acids (Table 3).The values for LOD and LQ are described in Table 3.The results of accuracy, intra and inter-assay obtained in the three different concentrations are described in Table 4.The recovery presented values between (87.9 ± 5.6)% and (112.3 ± 6.1)%.The results were reported in Table 5.

Determination of reference intervals
Figure 2 illustrates the distribution of the control group by gender and age.Table 6 shows the reference ranges calculated for all amino acids studied.

Method validation
The sample preparation is simple and fast, but should not be reinjected into the chromatograph since the reaction of amino acids with OPA is rather unstable and these compounds can be rapidly converted into non-fluorescent degradation products (18) .The disadvantage of using OPA consists in the instability of the fluorescent product and in its restriction of only reacting with primary amino acids.This is counterbalanced by the fact that its use allows high sensitivity and reproducible analysis (19) and its relatively low cost, making OPA a widely used derivatization reagent for the analysis of amino acid (18,20) .
The procedure was considered long for a laboratory routine.That occurs because, although the use of solid core column allowed excellent separation and good durability, when it is introduced into HPLC it requires a lower flow rate for not reaching high pressures.
The amino acids glycine and threonine were not included in the validation since they do not have good chromatographic separation.This can be explained by the fact that the retention time of both amino acids were subjected to the gradient of mobile phases A (32.0%) and B (68.0%).
Under the conditions tested, the gradient, that it is a mixture of mobile phases of different compositions, provided the coelution of threonine and glycine, favored the time for the chromatographic run and did not impair the determination and the reliability of other validated amino acids.
The results of the linearity limits of detection and quantification method were satisfactory.
The greatest variation found in the intra-assay (12.3%) was attributed to aspartate and can be explained by the lower concentration level for this amino acid (10 µmol/l).Comparing the results obtained in the intra-assay precision with the biological variation (21) described in Table 4, it is observed that all variations found in this study were lower than the established biological variation.
In the inter-assay, glutamine showed the greatest variation (15.2%-concentration of 180 µmol/l).This variation found was even higher than the within-subject biologic variation (21) described in Table 4.The variability attributed to glutamine may be connected to the gradient that starts exactly at the retention time of the glutamine, thus influencing the increased variability.Fekkes  (1996) assessed the inter-assay precision (n = 12) using HPLC and OPA-derivatization, the author also noted that the amino acid glutamine presented the greatest variation (25%) in the study.
For other amino acids, all variations in the inter-assay precision were less than the biological variation contained in Table 4.

Determination of reference intervals
; e CVg: coefficient of variation between-subject biological variation (21) .(1997) (10) due to lack of descriptive data, such as normality test, mean, median, minimum and maximum, necessary to statistical analysis.No appropriate study describing reference intervals for amino acids in children was found in national literature to comparison.

CONCLusION
The validated method demonstrated satisfactory linearity, precision, recovery, limits of quantification and detection, and it can be applied in the routine clinical practice.The study suggests that each laboratory should adopt its own reference intervals for amino acids in plasma.Even so, if this is not possible, the described reference

TAbLE 1 −
Solubility of the amino acids analyzed

TAbLE 2 −
Mobile phase gradient established by amino acid analysis

TAbLE 3 −
Linearity, limit of detection and quantification of the method r 2a : coefficient of determination; LOD b : limit of detection; LQ c : limit of quantification.

TAbLE 6 −
Reference ranges for plasma amino acids Amino acids (nmol/ml) methodology.The same occurred with the study ofLepage et al.