Target metabolic profiling analysis of free amino acids in plasma as EOC/TBDMS derivatives by GC-SIM-MS

Metabolic profiling analysis of free amino acids (AAs) in plasma (20 microL) was performed by gas chromatography-mass spectrometry in selected ion monitoring mode after ethoxycarbonyl/tert-butyldimethylsilyl derivatives. Characteristic fragment ions, including [M - 57](+) ions, permitted sensitive and selective detection of most of the AAs in the presence of co-extracted carboxylic acids, including free fatty acids, at much higher levels. The overall method was linear (r > or = 0.9991), reproducible (relative standard deviation = 2.3-8.8%) and accurate (relative error = -7.3-7.7%) with detection limits of 0.01-1.9 ng/mL. A total of 18 AAs, 15 protein AAs and three nonprotein AAs were quantitatively screened in a normal human plasma sample. This selective and simple method using a minimal sample volume was effective for the quantitation of plasma free AAs.     

HPLC-UV assay for monitoring total and unbound mycophenolic acid concentrations in children

A simple, accurate and sensitive HPLC method was developed for measuring total and unbound mycophenolic acid (MPA) in human plasma. Total MPA was extracted by protein precipitation and ultrafiltration was used to assess unbound MPA concentrations. The supernatant (20 microL) or ultrafiltrate (100 microL) was injected onto a C(18) HPLC column with a mobile phase of 0.05 m sodium phosphate buffer (pH 2.31)-acetonitrile (55:45, v/v for total MPA; 50:50 for unbound MPA) with UV detection at 254 nm. The extraction recovery was over 93% and reproducible. The assay was linear over the concentration range of 0.07-50 mg/L for total MPA and 4-1500 microg/L for unbound MPA. Intra- and inter-day assay reproducibility was less than 10%. Detection limits were 0.04 mg/L and 2 microg/L for total and unbound MPA, respectively. The assay utility was established in samples collected from five paediatric bone marrow transplant recipients who were receiving intravenous doses of mycophenolate mofetil. In these patients MPA concentrations ranged from 0.07 to 7.83 mg/L and unbound drug concentrations ranged from 2.1 to 107.5 microg/L. This method can be effectively applied to MPA pharmacokinetics in paediatric patients.     

Quantitative analysis of betulinic acid in mouse,rat and dog plasma using electrospray liquid chromatography/mass spectrometry

Betulinic acid is under development as a therapeutic agent for the treatment of metastatic malignant melanoma. In support of pharmacokinetic and toxicological evaluations, a robust assay based on liquid chromatography/mass spectrometry (LC/MS) was developed for the quantitative analysis of betulinic acid. Sample preparation consisted of deproteinization of the plasma by the addition of three volumes of acetonitrile and one volume of methanol followed by centrifugation. Aliquots of the supernatant were analyzed using an isocratic reversed-phase high-performance liquid chromatography (HPLC) system coupled to a negative ion electrospray mass spectrometer. Deprotonated molecules of betulinic acid and the isomeric internal standard oleanolic acid were detected using selected ion monitoring at m/z 455. The limit of detection of betulinic acid was 0.5 pg (1.1 fM) injected on-column (50 pg/mL, 10 microL injection volume), and the limit of quantitation was 2 pg (4.4 fM, 200 pg/mL, 10 microL injection volume). Betulinic acid was stable in plasma samples at -20 degrees C for at least 3 weeks. The intra-day and inter-day coefficients of variation of the assay were < or =6.4 and < or =9.0%, respectively. The utility of the assay was demonstrated by analyzing betulinic acid spiked into mouse, rat and dog plasma, by determining the extent of binding of betulinic acid to plasma proteins, and by measuring betulinic acid in mouse and rat plasma following intraperitoneal or intravenous administration in vivo. At 15 and 25 microg/mL in mouse, rat or dog plasma, betulinic acid was 99.99% bound to serum proteins, and, at 5 microg/mL, betulinic acid was > or =99.97% bound.     

Direct amino acid analysis method for speciation of selenoamino acids using high-performance anion-exchange chromatography coupled with integrated pulsed amperometric detection

Speciation analysis of selenomethylcysteine (SeMeCys), selenomethionine (SeMet) and selenocystine (SeCys) has been performed using a direct amino acid analysis method with high-performance anion-exchange chromatography (HPAEC) coupled with integrated pulsed amperometric detection (IPAD). Three selenoamino acids could be baseline-separated from 19 amino acids using gradient elution conditions for amino acids and determined under new six-potential waveform. Detection limits for SeMeCys, SeMet and SeCys were 0.25, 1 and 20 microg/L (25 microL injection, 10 times of the baseline noise), respectively. The relative standard deviations (RSDs) of 200 microg/L SeMeCys, SeMet and SeCys were 3.1, 4.1 and 2.8%, respectively (n=9, 25 microL injection). The proposed method has been applied for determination of selenoamino acids in extracts of garlic and selenious yeast granule samples. No selenoamino acids were found in garlic. Both SeMet and SeCys were detected in selenious yeast tablet with the content of 45 and 129 microg Se/g, respectively. Selenoamino acids standards were spiked in garlic and yeast granule samples and the recovery ranged from 90 to 106%.     

High-performance liquid chromatography-electrospray ionization mass spectrometric determination of nisoldipine in human plasma

A selective and sensitive high-performance liquid chromatography-electrospray ionization mass spectrometry (MS) for the determination of nisoldipine in human plasma is first presented. With nimodipine as the internal standard, nisoldipine is extracted from plasma with ethyl acetate. The organic layer is evaporated and the residue is resuspended in the mobile phase of methanol-water (80:20, v/v). An aliquot of 40 microL is chromatographically analyzed on an Agilent ODS C18 reversed-phase column (5 microm, 250- x 4.6-mm i.d.) by means of selected-ion monitoring mode MS. The calibration curve of nisoldipine in plasma exhibits a linear range from 0.5 to 20.0 ng/mL with a correlation coefficient of 0.9995. The limit of detection is 0.2 ng/mL. The within- and between-day variations (relative standard deviation) are less than 9.28% and 11.13% (n = 5), respectively. The developed method is validated through successful use for the analysis of nisoldipine contained in biological fluids resulting from a phase-I human pharmacokinetic study.     

Simultaneous determination of mycophenolic acid and valproic acid based on derivatization by high-performance liquid chromatography with fluorescence detection

A reliable and validated reversed-phase high-performance liquid chromatography (HPLC) method using fluorescence detection is reported for the simultaneous quantitation of mycophenolic acid (MPA) and valproic acid (VPA) in human plasma. The method is based on the pre-column derivatization of valproic acid with 4-bromomethyl-6, 7-dimethoxycoumarin (BrMMC) and online solvatochromism of MPA by pH adjustment. The linear calibration range was 0.50-30 microg/mL for MPA and 5.00-150 microg/mL for VPA. The relative standard deviations of the method of intra- and inter-day analyses (n = 6) were below 6.5 and 6.7% for MPA, and 5.8 and 6.3% for VPA, respectively. Dichloromethane was used for the simultaneous extraction of MPA and VPA from acidified plasma. This reliable method can be applied in the analysis of MPA and VPA in human plasma using only a small volume (100 microL).     

Development of a validated HPLC method for the determination of four penicillin antibiotics in pharmaceuticals and human biological fluids

A quantitative method for the determination of four penicillin antibiotics, amoxicillin (AMO), oxacillin (OXA), cloxacillin (CLO), and dicloxacillin (DICLO), has been developed. Separation was achieved on an Inertsil ODS-3 (250 x 4 mm, 5 microm) column after selective extraction of penicillin drugs from biological matrices by means of SPE. Gradient elution with a mobile phase consisting of 0.1% TFA (pH 1) and ACN, and PDA detection with monitoring at 240 nm was applied. Salicylic acid (5 ng/microL) was used as the internal standard. RP-8 Adsorbex Merck cartridges provided high absolute recoveries (98-101%). The developed method was fully validated in terms of selectivity, linearity, accuracy, precision, stability, and sensitivity. Repeatability (n = 8) and between-day precision (n = 8) revealed RSD <10%. Recoveries from biological samples ranged from 91 to 103%. The detection limits were estimated as 3.3 ng for AMO, OXA, and CLO, and 6.6 for DICLO in blood plasma. LOD in whole blood and urine was 6.6 ng. Injection volume was 20 microL. The method was applied to commercially available AMO containing pharmaceuticals and spiked biological matrices. The method was also applied to biological samples after AMO oral administration, where the drug was successfully identified and quantified.     

阴离子交换离子色谱法测定乳酸催化制备的丙烯酸

通过乳酸催化脱水制备丙烯酸具有良好的应用前景。为了对其中的催化过程进行有效、及时的监控,建立了一种同时测定乳酸及丙烯酸的阴离子交换色谱法(AEC)。选择Metrohm A Supp 5阴离子交换柱(150 mm×4.0 mm),以2 mmol/L Na2CO3+2 mmol/L NaHCO3混合水溶液为流动相,采用化学抑制电导检测技术,乳酸和丙烯酸在6 min内即可实现完全分离。乳酸和丙烯酸工作曲线的线性范围分别为0.1～500 mg/L和0.1～200 mg/L,检出限分别为0.030 mg/L和0.035 mg/L,加标回收率分别为100.7%～106%和99.6%～103%,相对标准偏差分别为2.16%~2.49%和2.42%~2.48%。该方法准确、快速、灵敏、重现性好。     

高效液相色谱法测定吲哚美辛控释胶囊的血药浓度和生物利用度

采用ＯＤＳ柱,甲醇－稀磷酸溶液（７６２４）为流动相,２６０ｎｍ为检测波长,建立了测定血浆中吲哚美辛浓度的高效液相色谱法,并测定了吲哚美辛控释胶囊炎痛康的血药浓度。结果表明,血浆中吲哚美辛浓度在０．１２５～５．０ｍｇ／Ｌ范围内线性关系良好（ｒ＝０．９９９６）,检测限６２．５μｇ／Ｌ（Ｓ／Ｎ＝３１）,平均回收率为１００．４％,日内和日间ＲＳＤ均小于５％。１１位受试者单剂量口服炎痛康后的相对生物利用度为１０２．３８％。     

LC-MS/MS determination of 2-(4-((2-(2S,5R)-2-Cyano-5-ethynyl-1-pyrrolidinyl)-2-oxoethylamino)-4-methyl-1-piperidinyl)-4-pyridinecarboxylic acid (ABT-279) in dog plasma with high-throughput protein precipitation sample preparation

As an effective DPP-IV inhibitor, 2-(4-((2-(2S,5R)-2-Cyano-5-ethynyl-1-pyrrolidinyl)-2-oxoethylamino)-4-methyl-1-piperidinyl)-4-pyridinecarboxylic acid (ABT-279), is an investigational drug candidate under development at Abbott Laboratories for potential treatment of type 2 diabetes. In order to support the development of ABT-279, multiple analytical methods for an accurate, precise and selective concentration determination of ABT-279 in different matrices were developed and validated in accordance with the US Food and Drug Administration Guidance on Bioanalytical Method Validation. The analytical method for ABT-279 in dog plasma was validated in parallel to other validations for ABT-279 determination in different matrices. In order to shorten the sample preparation time and increase method precision, an automated multi-channel liquid handler was used to perform high-throughput protein precipitation and all other liquid transfers. The separation was performed through a Waters YMC ODS-AQ column (2.0 x 150 mm, 5 microm, 120 A) with a mobile phase of 20 mm ammonium acetate in 20% acetonitrile at a flow rate of 0.3 mL/min. Data collection started at 2.2 min and continued for 2.0 min. The validated linear dynamic range in dog plasma was between 3.05 and 2033.64 ng/mL using a 50 microL sample volume. The achieved r(2) coefficient of determination from three consecutive runs was between 0.998625 and 0.999085. The mean bias was between -4.1 and 4.3% for all calibration standards including lower limit of quantitation. The mean bias was between -8.0 and 0.4% for the quality control samples. The precision, expressed as a coefficient of variation (CV), was < or =4.1% for all levels of quality control samples. The validation results demonstrated that the high-throughput method was accurate, precise and selective for the determination of ABT-279 in dog plasma. The validated method was also employed to support two toxicology studies. The passing rate was 100% for all 49 runs from one validation study and two toxicology studies.     

A rapid and sensitive high-performance liquid chromatography assay for rofecoxib in human serum

Rofecoxib is a selective cyclooxygenase (COX)-2 inhibitor that is approved for the treatment of acute pain and osteoarthritis in adults. A sensitive and rapid high-performance liquid chromatographic (HPLC) method of determining rofecoxib in human serum is described. Alkalinized plasma samples are extracted into an organic solvent containing an internal standard and evaporated under nitrogen. The dried sample residues are reconstituted with mobile phase and analyzed by HPLC. The method uses 100 microL of the sample and is linear from 20 to 2000 ng/mL of rofecoxib. Precision and accuracy studies are performed. Stability of the drug in serum over four weeks is documented. This new method is simple, sensitive, precise, and accurate. Its use will translate into faster laboratory turnaround time, and the small sample volume required (100 microL) makes this assay suitable for pediatric patients. This assay will expedite pharmacokinetic studies and the therapeutic drug monitoring of rofecoxib and possibly other COX-2 inhibitors.     

Quantitative high-throughput determination of endogenous retinoids in human plasma using triple-stage liquid chromatography/tandem mass spectrometry

A high-throughput ultrasensitive analytical method based on liquid chromatography with positive ion atmospheric pressure chemical ionization (APCI) coupled to tandem mass spectrometric detection (LC/MS/MS) was developed for the determination of all-trans-4-oxo-retinoic acid (at4oxoRA), 13-cis-4-oxo-retinoic acid (13c4oxoRA), 13-cis-retinoic acid (13cRA), all-trans-retinoic acid (atRA) and all-trans-retinol (atROH) in human plasma. A stable isotope of atRA was used as internal standard (IS). The analytes and IS were isolated from 100 microL plasma by acetonitrile mono-phase extraction (MPE) performed in black 96-well microtiterplates. A 100 microL injection was focused on-column and chromatographed on an Agilent ZORBAX SB-C18 rapid-resolution high-throughput (RRHT) column with 1.8-microm particles (4.6 mmx50 mm) maintained at 60 degrees C. The initial mobile phase composition was acetonitrile/water/formic acid (10:90:0.1, v/v/v) delivered at 1.8 mL/min. Elution was accomplished by a fast gradient to acetonitrile/methanol/formic acid (90:10:0.1, v/v/v). The method had a chromatographic total run time of 7 min. An Applied Biosystems 4000 Q TRAP linear tandem mass spectrometer equipped with a heated nebulizer (APCI) ionization source was operated in multiple reaction monitoring (MRM) mode with the precursor-to-product ion transitions m/z 315.4-->297 (4-oxo-retinoic acids), 301.2-->205 (retinoic acids), 305.0-->209 (IS) and 269.2-->93 (retinol) used for quantification. The assay was fully validated and found to have acceptable accuracy, precision, linearity, sensitivity and selectivity. The mean extraction recoveries from spiked plasma samples were 80-105% for the various retinoids at three different levels. The intra-day accuracy of the assay was within 8% of nominal and intra-day precision was better than 8% coefficient of variance (CV) for retinoic acids. Inter-day precision results for quality control samples run over a 12-day period alongside clinical samples showed mean precision better than 12.5% CV. The limit of quantification was in the range of 0.1-0.2 ng/mL and the mass limit of detection (mLOD) was in the range 1-4 pg on column for the retinoic acids. The assay has been successfully applied to the analysis of 1700 plasma samples.     

Determination of tartaric acid in wines by FIA with tubular tartrate-selective electrodes

A flow injection analysis (FIA) system comprising a tartrate-(TAT) selective electrode has been developed for determination of tartaric acid in wines. Several electrodes constructed for this purpose had a PVC membrane with a complex of quaternary ammonium and TAT as anion exchanger, a phenol derivative as additive, and a more or less polar mediator solvent. Characterization of the electrodes showed behavior was best for membranes with o-nitrophenyl octyl ether as solvent. On injection of 500 microL into a phosphate buffer carrier (pH = 3.1; ionic strength 10(-2) mol/L) flowing at 3 mL/min, the slope was 58.06 +/- 0.6 with a lower limit of linear range of 5.0 x 10(-4) mol/L TAT and R2 = 0.9989. The interference of several species, e.g. chloride, bromide, iodide, nitrate, gallic acid, tannin, sucrose, glucose, fructose, acetate, and citrate, was evaluated in terms of potentiometric selectivity coefficients. The Hofmeister series was followed for inorganic species and the most interfering organic ion was citrate. When red and white wines were analyzed and the results compared with those from an independent method they were found to be accurate, with relative standard deviations below 5.0%.     

高效液相色谱法同时测定生物质乳酸发酵液中有机酸及糖类化合物

建立了高效液相色谱(HPLC)同时测定生物质乳酸发酵液中有机酸及糖类的分析方法。使用Bio-Rad Aminex HPX-87H色谱柱,以5 mmol/L的H2SO4为流动相,在柱温55 ℃,流速0.6 mL/min条件下,采用示差折光检测器进行检测。结果表明,该方法可在17 min内实现发酵液中各种有机酸和糖类化合物等的完全分离与定量,6种有机酸和3种糖类化合物在0.15～5.19 g/L范围内的线性关系良好,回归方程的线性相关系数在0.9998以上。将该法用于米根霉发酵液的检测,两个水平的加标回收率为96.91%～103.11%,相对标准偏差(n=6)为0.81%～4.61%。该法适用于微生物发酵液中多种有机酸和糖类的快速、高效分离和定量测定。     

Determination of molybdenum by atomic-absorption spectrometry after separation by 5,5'-methylenedisalicylohydroxamic acid extraction and further reaction with thiocyanate and tin (II)

A method is described for the determination of molybdenum down to the microgram level, in samples of soil, steels, fertilizers and pharmaceuticals. After attack with acids, this element is separated from matrix elements by extraction of its 5,5'-methylenedisalicylohydroxamic acid (MEDSHA) complex from 4M hydrochloric acid, into methyl isobutyl ketone. Molybdenum is determined by atomic-absorption spectrometry (AAS), after conversion of the Mo-MEDSHA complex into the MoSCN(-) complex in the organic phase. The detection limit is 0.03 microg/ml, with a relative standard deviation not exceeding 1.5% at a level of 2 microg/ml. The method is highly selective and suffers only from interference by tungsten.     

Determination of organic acid impurities in lactic acid obtained by fermentation of sugarcane juice

Lactic acid produced by fermentation process mostly contains a number of aliphatic carboxylic acids as impurities. In this work, carboxylic acid impurities in lactic acid samples from a number of sources were determined at ppm levels. A simple HPLC method was developed that utilized a new generation polar embedded reverse phase, 20mM phosphate buffer at pH 2.20 (±0.05) and UV detection at 210 nm. The method enabled quantitative analysis of the above acids in lactic acid matrix. The experimental conditions for column temperature, mobile phase pH and flow rate were optimized. A detailed validation of the method was performed for linearity, precision, accuracy, selectivity, limit of detection (LOD), limit of quantitation (LOQ), ruggedness and repeatability and reproducibility (R&R).     

Quantification of 3-nitrotyrosine in biological tissues and fluids: Generating valid results by eliminating artifactual formation

Reactive nitrogen species such as peroxynitrite can nitrate specific amino acids, whether free or protein bound, and 3-nitrotyrosine is believed to be one marker of this reaction. To examine the significance of this pathway in biological systems we have developed an accurate, sensitive, and specific assay for 3-nitrotyrosine based on combined liquid chromatography tandem mass spectrometry. Our approach allowed simultaneous analysis of both tyrosine and 3-nitrotyrosine and employs isotopomer standards (i.e., [15N1, 13C9]-tyrosine and [13C6]-3nitrotyrosine). Calibration curves were linear (r2 = 0.999) across the range 0.5-100 pg/microL (i.e., 2.2-442 fmol/microL), and the detection limit for standard samples was 0.5 pg/microL (2.2 fmol/microL, or 10 fmol on column; S/N = 5) or 1 pg/microL (4.4 fmol/microL) for extracted (biological) samples. As a component of this study we have undertaken an extensive investigation of artifactual formation of 3-nitrotyrosine under conditions that exist during sample extraction and derivatization. Our studies show that under appropriate conditions (low pH, elevated temperatures, and in the presence of a vast excess of the two substrates, tyrosine and the nitrate anion), 3-nitrotyrosine can readily be formed as an artifact.     

A rapid HPLC method with fluorometric detection for determination of plasma itraconazole and hydroxy-itraconazole concentrations in cystic fibrosis children with allergic bronchopulmonary aspergillosis

The development and validation of a simple, rapid and selective high-performance liquid chromatography (HPLC) method is described for the quantitation of itraconazole and hydroxy-itraconazole in 100 microL of plasma from a paediatric population. The mobile phase of methanol (75% v/v) and water (25% v/v) was pumped at 1 mL/min through a C18 Symmetry (3.9 mm i.d. x 150 mm) cartridge. Using a protein-precipitation method, 100 microL internal standard (IS) solution (R051012, 555 microg/L in acetonitrile) were added to 100 microL of plasma followed by 10 microL zinc sulphate solution (20% w/v). Itraconazole, hydroxy-itraconazole and IS eluted at 4.7, 8.3 and 12.5 min, respectively and were detected fluorometrically at 250 nm (excitation) and 380 nm (emission). Recoveries were 87.1-96.7%. Calibrations in drug-free plasma were linear (r2 > 0.99) from 50 to 2000 microg/L, using 1/c2 (c = concentration) weighting. Intraday and interday imprecision (CV%) was 4.8-17.3 and 6.3-16.6% for itraconazole, and 4.6-17.9 and 7.02-18.4% for hydroxy-itraconazole. Inaccuracy was -7.1 to -14.7% for itraconazole and -0.1 to -9.7% for hydroxy-itraconazole. The clinical application of this method was demonstrated by measurement of itraconazole and hydroxy-itraconazole in plasma samples drawn from paediatric cystic fibrosis patients, who were prescribed itraconazole for treatment of allergic bronchopulmonary aspergillosis.     

Determination on the microscale of plasmatic lactic acid as its t-butyldimethylsilyl derivative by stable-isotope dilution using capillary gas chromatography/mass spectrometry.

A new sensitive and precise method for the determination of lactic acid in plasmatic microsamples (50 microL) has been developed. Lactic acid was directly extracted from plasma by ethyl acetate in acidic conditions, and analysed as its di-t-butyldimethylsilyl derivative by capillary gas chromatography/electron-impact mass spectrometry (GC/MS). The internal standard was a previously synthesized deuterated compound, 3-[2H]-(2R)-lactic acid. The method gives good reproductibility and precision, the overall standard deviation being better than 3%. The GC/MS assay was in good agreement with the enzymatic determination.     

Simultaneous determination of simvastatin and simvastatin acid in human plasma by LC-MS/MS without polarity switch: application to a bioequivalence study

A simple, specific and sensitive LC-MS/MS assay for simultaneous determination of simvastatin (SV) and its active beta-hydroxy acid metabolite, simvastatin acid (SVA) in human plasma was developed using a statin analog as internal standard (IS). The method was validated over a dynamic linear range of 0.20-100.00 ng/mL for SV and 0.10-50.00 ng/mL for SVA with correlation coefficient r > or = 0.9987 and 0.9989, respectively. The analytes and IS were extracted from 500 microL aliquots of human plasma via liquid-liquid extraction using methyl tert-butyl ether and separated through an Aquasil C18 column (100 mm x 2.1 mm, 5 microm). Detection of analytes and IS was done by MS/MS with a turbo ion spray interface operating in positive ion and selective reaction monitoring acquisition mode. The total chromatographic run time was 3.0 min. Flash freezing of the aqueous phase was an added advantage during liquid-liquid extraction, which considerably reduced time and labour. The method was extensively validated for its accuracy, precision, recovery, stability studies and matrix effect. The method was successfully used for bioequivalence study of 40 mg SV tablet formulation in 12 human subjects under fasting condition.