Determination of nitrate in biological fluids by HPLC

Nitrate is the stable product of nitric oxide, which is physiologically active radical, an immunomodulator and a neuromodulator; its quantification in biological fluids is important to study the physiological and biochemical nature. Therefore, the purpose of this study was to quantify nitrate in different biological fluids like serum, cerebrospinal fluid [CSF] and ascetic fluid [ASF] using HPLC technique. A new HPLC method for the estimation of nitrate in serum, CSF and ASF was developed using the mobile phase of 1.0mM each of Na[2]CO[3] and NaHCO3 [1:1, v/v, pH 5 with H[3]PO[4]] at a flow rate of 1.0mLmin[-1]. Eluate was detected at 220nm with the retention time of nitrate 2.55 min. The LOD and LOQ values of nitrate were 0.03 micro gmL[-1] and 0.098 micro gmL[-1], respectively. Nitrate was eluted through SAX Hypersil column of 150 x 4.6mm, id, 5 micro m particle size. Run time was 10min. The method was validated according to the FDA guidelines and was found linear in the range of 0.39 to 50 micro gmL[-1] and CV was <3%, within limits of FDA guidelines. The method was used successfully for the estimation of nitrate in biological fluids like serum, CSF and ASF of 20 patients each. This is an alternate and reproducible method for the detection of nitrates in biological fluids

Liquid Chromatography-Mass Spectrometry-Based In Vitro Metabolic Profiling Reveals Altered Enzyme Expressions in Eicosanoid Metabolism

BACKGROUND: Eicosanoids are metabolites of arachidonic acid that are rapidly biosynthesized and degraded during inflammation, and their metabolic changes reveal altered enzyme expression following drug treatment. We developed an eicosanoid profiling method and evaluated their changes on drug treatment. METHODS: Simultaneous quantitative profiling of 32 eicosanoids in liver S9 fractions obtained from rabbits with carrageenan-induced inflammation was performed and validated by liquid chromatography-mass spectrometry coupled to anion-exchange solid-phase purification. RESULTS: The limit of quantification for the devised method ranged from 0.5 to 20.0 ng/mg protein, and calibration linearity was achieved (R 2>0.99). The precision (% CV) and accuracy (% bias) ranged from 4.7 to 10.3% and 88.4 to 110.9%, respectively, and overall recoveries ranged from 58.0 to 105.3%. Our method was then applied and showed that epitestosterone treatment reduced the levels of all eicosanoids that were generated by cyclooxygenases and lipoxygenases. CONCLUSIONS: Quantitative eicosanoid profiling combined with in vitro metabolic assays may be useful for evaluating metabolic changes affected by drugs during eicosanoid metabolism.

Dried Blood Spot Testing for Seven Steroids Using Liquid Chromatography-Tandem Mass Spectrometry With Reference Interval Determination in the Korean Population

BACKGROUND: Conventional screening for congenital adrenal hyperplasia (CAH) using immunoassays generates a large number of false-positive results. A more specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been introduced to minimize unnecessary follow-ups. However, because of limited data on its use in the Korean population, LC-MS/MS has not yet been incorporated into newborn screening programs in this region. The present study aims to develop and validate an LC-MS/MS method for the simultaneous determination of seven steroids in dried blood spots (DBS) for CAH screening, and to define age-specific reference intervals in the Korean population. METHODS: We developed and validated an LC-MS/MS method to determine the reference intervals of cortisol, 17-hydroxyprogesterone, 11-deoxycortisol, 21-deoxycortisol, androstenedione, corticosterone, and 11-deoxycorticosterone simultaneously in 453 DBS samples. The samples were from Korean subjects stratified by age group (78 full-term neonates, 76 premature neonates, 89 children, and 100 adults). RESULTS: The accuracy, precision, matrix effects, and extraction recovery were satisfactory for all the steroids at three concentrations; values of intra- and inter-day precision coefficients of variance, bias, and recovery were 0.7-7.7%, -1.5-9.8%, and 49.3-97.5%, respectively. The linearity range was 1-100 ng/mL for cortisol and 0.5-50 ng/mL for other steroids (R2>0.99). The reference intervals were in agreement with the previous reports. CONCLUSIONS: This LC-MS/MS method and the reference intervals validated in the Korean population can be successfully applied to analyze seven steroids in DBS for the diagnosis of CAH.

Quantification of Human Plasma-Busulfan Concentration by Liquid Chromatography-Tandem Mass Spectrometry

BACKGROUND: Busulfan, an alkylating agent administered prior to hematopoietic stem cell transplantation, has a narrow therapeutic range and wide variability in metabolism. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for rapid and accurate quantification of plasma busulfan. METHODS: Busulfan was separated and detected using an LC system containing a C18 column equipped with MS/MS. The sample was eluted with a mobile phase gradient for a total run time of 10 min. Plasma busulfan concentration was quantified against a 6-point standard curve in a multiple reaction monitoring mode at mass-to-charge (m/z) 264.1 > 151.1. Precision, recovery, matrix effect, linearity, detection capability, carryover, and stability were evaluated. The range of plasma busulfan concentration was obtained by analyzing samples from 9 children receiving busulfan. RESULTS: The coefficients of variation of within-run and within-laboratory precision were all below 5%. Recoveries were all within the range of 100-105%. Linearity was verified from 0 to 5,000 ng/mL. Limit of detection and limit of quantification were 1.56 and 25 ng/mL, respectively. Carryover rate was within allowable limits. Plasma busulfan concentration was stable for 2 weeks at -20degrees C and -80degrees C, but decreased by 25% when the plasma was stored for 24 hr at room temperature, and by <5% in 24 hr at 4degrees C. The plasma busulfan concentrations were between 347 ng/mL and 5,076 ng/mL. CONCLUSIONS: Our method using LC-MS/MS enables highly accurate, reproducible, and rapid busulfan monitoring with minimal sample preparation. The method may also enable safe and proper dosage.

Assessment of the Trueness and Inter-Laboratory Precision of Routine Uric Acid Assays Using 4 Frozen Pooled Serum Samples Measured by the Japan Society of Clinical Chemistry's HPLC Method

BACKGROUND: Reference procedures are required for evaluating the accuracy of routine analytical systems for uric acid (UA). External quality assessment (EQA) for UA has only been conducted with quality controls in China, and the results have not been published. This study was designed to investigate both the trueness and inter-laboratory precision of UA measurements among routine analytical systems using a candidate reference method. METHODS: We performed the HPLC method recommended by the Japan Society of Clinical Chemistry (JSCC). Next, we evaluated its analytical performance and validated its trueness. The performance of 4 routine analytical systems (5 instruments per system, n=20) for UA was assessed by using 4 frozen pooled serum samples measured by the HPLC method according to biologically relevant quality goals. RESULTS: Within-run, between-run, inter-day, and total CV of the method were less than 0.3%, 0.4%, 1.8%, and 2.6%, respectively. The UA measurements were consistent with the target values of standard reference material (SRM) 909b, the sixth ring trial for Reference Laboratories (RELA-2008) specimen, and national primary reference materials. The 4 frozen pooled serum samples were homogeneous, stable, and commutable. All routine systems achieved the desirable performance goal (total error <11.9%). CONCLUSIONS: We successfully reproduced the JSCC's HPLC method, which was simple, specific, precise, and accurate. We recommend this method as a reference method for UA measurement in human serum. Four routine analytical systems for UA measurement had acceptable traceability, and their UA results showed good concordance.

RP-HPLC analytical method development and optimization for quantification of donepezil hydrochloride in orally disintegrating tablet

An easy, fast and validated RV-HPLC method was invented to quantify donepezil hydrochloride in drug solution and orally disintegrating tablet. The separation was carried out using reversed phase C-18 column [Agilent Eclipse Plus C-18] with UV detection at 268 nm. Method optimization was tested using various composition of organic solvent. The mobile phase comprised of phosphate buffer [0.01M], methanol and acetonitrile [50:30:20, v/v] adjusted to pH 2.7 with phosphoric acid [80%] was found as the optimum mobile phase. The method showed intraday precision and accuracy in the range of 0.24% to -1.83% and -1.83% to 1.99% respectively, while interday precision and accuracy ranged between 1.41% to 1.81% and 0.11% to 1.90% respectively. The standard calibration curve was linear from 0.125 micro g/mL to 16 micro g/mL, with correlation coefficient of 0.9997 +/- 0.00016. The drug solution was stable under room temperature at least for 6 hours. System suitability studies were done. The average plate count was > 2000, tailing factor <1, and capacity factor of 3.30. The retention time was 5.6 min. The HPLC method was used to assay donepezil hydrochloride in tablet and dissolution study of in-house manufactured donepezil orally disintegrating tablet and original Aricept

Validity of Glycated Hemoglobin in Screening and Diagnosing Type 2 Diabetes Mellitus in Chinese Subjects

BACKGROUND/AIMS: The application of glycated hemoglobin (HbA1c) for the diagnosis of diabetes is currently under extensive discussion. In this study, we explored the validity of using HbA1c as a screening and diagnostic test in Chinese subjects recruited in Nanjing, China. METHODS: In total, 497 subjects (361 men and 136 women) with fasting plasma glucose (PG) > or = 5.6 mmol/L were recruited to undergo the oral glucose tolerance test (OGTT) and HbA1c test. Plasma lipid, uric acid, and blood pressure were also measured. RESULTS: Using a receiver operating characteristic curve, the optimal cutoff point of HbA1c related to diabetes diagnosed by the OGTT was 6.3%, with a sensitivity and specificity of 79.6% and 82.2%, respectively, and the area under the curve was 0.87 (95% confidence interval, 0.83 to 0.92). A HbA1c level of 6.5% had a sensitivity and specificity of 62.7% and 93.5%, respectively. When comparing the HbA1c > or = 6.5% or OGTT methods for diagnosing diabetes, the former group had significantly higher HbA1c levels and lower levels of fasting and 2-hour PG than the latter group. No significant difference was observed in the other metabolism indexes between the two groups. CONCLUSIONS: Our results suggest that HbA1c > or = 6.5% has reasonably good specificity for diagnosing diabetes in Chinese subjects, which is in concordance with the American Diabetes Association recommendations.

Separación de aminoácidos mediante cromatografía líquida de alta resolución / Amino acid analysis by high-performance liquid chromatography

Se presenta una compilación de la aplicación de la cromatografía líquida de alta resolución en el análisis de aminoácidos. Se analizan las formas de derivatización, se comparan los distintos derivados según sus alcances y limitaciones, la forma de detección y los distintos modos cromatográficos: cromatografía de intercambio iónico, cromatografía en fase normal, cromatografía en fase reversa y cromatografía en fase quiral. También se presentan los métodos automatizados comerciales más recientes para este tipo de análisis. Se dan ejemplos de aplicación de interés en el campo clínico-bioquímico, con el análisis de muestras de líquido cafalorraquídeo, de orina y de plasma/sangre o manchas de sangre seca. El análisis de los aminoácidos de hidrolizados de proteínas y de péptidos resulta más complejo y las condiciones de hidrólisis juegan un rol muy importante en los resultados, que son tratados en esta compilación. Se incluyen, además, aspectos referidos al análisis de los aminoácidos lábiles en proteínas, la preparación de las muestras, las recomendaciones prácticas al hacer HPLC y la influencia de los buffers