Simultaneous clinical monitoring of lactic acid, pyruvic acid and ketone bodies in plasma as methoxime/tert-butyldimethylsilyl derivatives by gas chromatography-mass spectrometry in selected ion monitoring mode

Simultaneous determination of lactic acid, pyruvic acid, 3-hydroxybutyric acid and acetoacetic acid for clinical monitoring of lactic acidosis and ketone body formation in human plasma (20 microL) was performed by gas chromatography-mass spectrometry in selected ion monitoring (SIM) mode after generating methoxime/tert-butyldimethylsilyl derivatives. All of the targeted carboxylic acids were detected by characteristic fragment ions, which permitted sensitive and selective identification in the presence of co-extracted free fatty acids and other acidic metabolites at much higher levels. The method was linear (r>or=0.9991), reproducible (% relative standard deviation=1.2-5.8), and accurate (% relative error=-7.2-7.6), with detection limits of 0.05-1.7 ng/mL. This rapid, accurate and selective method using minimal plasma samples (20 microL) is useful in the clinical monitoring of lactic acidosis and ketone body formation in plasma.     

Qualitative and quantitative analysis of amino acids by capillary electrophoresis-electrospray ionization-tandem mass spectrometry

We describe a method to identify and quantify amino acids using capillary electrophoresis-electrospray ionization-triple-quadrupole tandem mass spectrometry (CE-ESI-MS/MS). Amino acids, including physiological amino acids, were first separated by CE under acidic pH conditions and then detected by MS/MS. To efficiently introduce the whole sample into the capillary, no electrical potential was applied to the electrospray probe until running electrophoresis. The position of the electrosprayer with respect to the MS capillary entrance drastically affected sensitivity and generation of cluster ions. MS/MS with multiple reaction monitoring (MRM) detection was performed to obtain sufficient selectivity and sensitivity. Under optimized CE-MS/MS conditions, the minimum detectable levels for 32 free amino acids normally found in proteins and other physiological amino acids were between 0.1 and 14 micromol/L with pressure injection of 50 mbar for 3 s (3 nL) at a signal-to-noise ratio of 3. For most amino acids, this constitutes a severalfold increase in sensitivity compared to CE-MS. The relative standard deviations (% RSD) for all amino acids were better than 0.4% for migration times and between 1.4% and 8.6% for peak areas (n = 10). Since amino acids exhibited characteristic MS/MS spectra, this approach is useful for the simultaneous, selective, quantitative, and reproducible analysis of amino acids in physiological and biological samples that contain various kinds of matrices. The power of the method was demonstrated by analyzing amino acids in human urine.     

Improving detection sensitivity of amino acids in thyroid tissues by using phthalic acid as a mobile phase additive in hydrophilic interaction chromatography-electrospray ionization-tandem mass spectrometry

In this work, 0.08 mmol L⁻¹ of phthalic acid was introduced as a mobile phase additive to quantify free amino acids (AAs) by hydrophilic interaction liquid chromatography (HILIC) coupled with electrospray ionization tandem mass spectrometry (ESI-MS/MS). The addition of phthalic acid significantly increased the signal intensity of protonated AA ions, resulting from the decrease of the relative abundance of AA sodium adducts. Meanwhile, the chromatographic peak shapes of AAs were optimized. As a consequence, there was a noticeable increase in the sensitivity of detection for AAs. The limits of detection (LOD) and quantification (LOQ) of the AAs ranged from 0.0500 to 20.0 ng mL⁻¹ and from 0.100 to 50.0 ng mL⁻¹, respectively, which were 4–50 times lower compared to the values measured without the addition of phthalic acid. The enhanced detection and separation of AAs were obtained by merely adding phthalic acid to the mobile phase without changing other conditions. Eventually, this simple method was validated and successfully applied to the analysis of twenty-four kinds of free AAs in human thyroid carcinoma and para-carcinoma tissues, demonstrating a significant increase of most AAs in thyroid carcinoma tissues (p < 0.05).     

Development and validation of a high-performance liquid chromatography-electrospray ionization-mass spectrometry assay for the determination of zaleplon in human plasma

In this paper, a sensitive and rapid chromatographic procedure using a selective analytical detection method (electrospray ionization-mass spectrometry in selected-ion monitoring mode) in combination with a simple and efficient sample preparation step is first presented for the determination of zaleplon in human plasma. The separation of the analyte, internal standard, and possible endogenous compounds are accomplished on a phenomenex Luna 5-microm C8(2) column (250- x 4.6-mm i.d.) with methanol-water (75:25, v/v) as the mobile phase. In order to optimize the mass detection of zaleplon, several parameters such as ionization mode, fragmentor voltage, m/z ratios of ions monitored, type of organic modifier, and eluent additive in the mobile phase are discussed. An internal standard is selected to guarantee the quantitative accuracy. Each analysis takes less than 6 min. The calibration curve of zaleplon in the range of 0.1-60.0 ng/mL in plasma is linear with a correlation coefficient of > 0.9992, and the detection limit (s/n = 3) is 0.1 ng/mL. The within- and between-day variations (relative standard deviation) in the zaleplon plasma analysis are less than 2.4% (n = 15) and 4.7% (n = 15), respectively. The application of this method is demonstrated for the analysis of zeleplon plasma samples in a Phase-I human pharmacokinetic study.     

Simultaneous analysis of six aristolochic acids and five aristolactams in herbal plants and their preparations by high-performance liquid chromatography-diode array detection-fluorescence detection

Aristolochic acid analogues, including aristolochic acids (AAs) and aristolactams (ALs), are known to be nephrotoxic, carcinogenic and mutagenic. In this paper, a high-performance liquid chromatography-diode array detection-fluorescence detection (HPLC-DAD-FLD) method was developed for the simultaneous determination of six AAs together with five ALs. Baseline separation was obtained on an ODS C18 analytical column with 0.2% HAc/methanol gradient elution. The hyphenation of DAD and FLD allows the method to directly meet the analysis requirements of most herbal plants with high sensitivity and selectivity. For trace analysis, aristolochic acids were reduced to their corresponding aritstolactams in acidic solution containing iron powder, and then high sensitive detection and quantification were carried out. The method was successfully validated in the matrices of various Aristolochiaceae plants and their preparations. Linearities of around 3-4 orders of magnitude were obtained with correlation coefficients exceeding 0.9970. The detection limits were decreased to 0.2ng/ml. Satisfactory intra-day and inter-day precisions were achieved with RSDs less than 5.74%, and the average recovery factors were in the range of 94.5-99.2%.     

Determination of Proteinogenic Amino Acids in Human Plasma by Capillary Electrophoresis with Contactless Conductivity Detection

Capillary electrophoresis (CE) with contactless conductivity detection (CCD) represents a viable alternative to liquid chromatography in the analysis of amino acids (AA) in human plasma. After optimizing the composition of the background electrolyte (BGE), and introducing simple plasma pretreatment to remove spurious protein components, the CE/CCD methods allows determination of 18 from 20 proteinogenic AAs, three nonproteinogenic AAs and creatinine in 60 minutes. AAs are separated in their cationic forms in BGE containing 1.7 M acetic acid and 0.1% hydroxyethylcellulose, pH 2.2. LODs for individual AAs vary in an acceptable range with minimum of 4.3 μM for Arg and maximum of 42.9 μM for Cys. Mean concentrations and concentrations ranges for AAs in human plasma samples from 9 healthy individuals are found to agree well with those determined by an LC method in other two laboratories.     

二维阀切换离子色谱法测定海带中游离氨基酸

建立一种测定海带中游离氨基酸的阀切换高效阴离子交换色谱耦合脉冲安培检测器法。采用一根阳离子交换柱对氨基酸进行富集,而后经阀切换至氨基酸分析柱Amino Pac~PA-10(250 mm×2 mm)上分离并进入安培检测器检测。在最佳分离条件下,20种氨基酸的质量浓度在0.1~20.0 mg/L范围内与其色谱峰面积线性关系良好,线性相关系数r~20.99,20种氨基酸的检出限为0.01 mg/L,加标回收率为83.12%~117.34%,测定结果的相对标准偏为1.02%~13.05%(n=8)。该方法样品前处理简单,无基底杂质干扰,适用于海带样品中游离氨基酸的测定。     

Determination and pharmacokinetic study of nodakenin in rat plasma by RP-HPLC method

A simple, sensitive and selective RP-HPLC method has been developed for quantification of nodakenin in rat plasma. Nodakenin in rat plasma was extracted with acetonitrile, which also acted as a deproteinization agent. Chromatographic separation of nodakenin was performed on an analytical Diamonsil ODS C18 column, with a mobile phase of MeOH-H2O (1:1, v/v) at a flow-rate of 1.0 mL/min, and UV detection was set at 330 nm. The calibration curve was linear over the range 0.2-12.0 microg/mL (R2 = 0.9995) in rat plasma. The lower limit of detection and quantification were 0.01 and 0.1 microg/mL, respectively, using the rat plasma sample. The extraction recoveries were 77.36 +/- 4.56, 82.89 +/- 1.84 and 81.66 +/- 2.49% at concentrations of 1.0, 5.0 and 10.0 microg/mL, respectively. The intra- and inter-day precision and accuracy were validated by relative standard deviation and relative error, which were in the ranges 5.07-5.83 and 3.95-6.29%, respectively. After i.v. administration to rats at a single dose of 40 mg/kg, the plasma concentration-time curve of nodakenin was best conformed to a two-compartment open model. This assay method has been successfully applied to the study of the pharmacokinetics of nodakenin in rats.     

Establishing a sensitive capillary electrophoresis‐UV method for direct determination of amino acids to evaluate vinegar quality

In this study, the capillary electrophoresis method with ultraviolet detection was established to directly determine amino acids in vinegar, according to the coordination interaction between amino acids (AAs) and copper ions. The online sweeping technique was combined to improve the detection sensitivity. The quality of vinegar was evaluated with AAs as parameters by United Nations Food Agriculture Organization/Word Health Organization AAs model and principal component analysis. Optimum conditions were obtained under 50 mM CuSO₄ and adjusted pH 4.40 with 8 mM acetate, 70 s injection time, 22.5 kV separation voltage, and 254 nm detected wavelength. Method validation, indicating good linearity (R² > 0.9989), precision with an RSD less than 8.0% (n = 5), LOD (0.13–0.25 μg/mL), LOQ (0.43–0.83 μg/mL) and recovery (80.5–112.6%). Under the optimal conditions, AAs in vinegar can be directly separated which is propitious for the quality evaluation of vinegar.     

Online concentration of aristolochic acid I and II in Chinese medicine preparations by micellar electrokinetic chromatography

In this study, an online concentration method in micellar electrokinetic chromatography (MEKC) applying field-enhanced sample injection (FESI) mode was developed for the detection of aristolochic acids (AAs) in Chinese medicine preparations. AA-I and AA-II were baseline separated with high separation efficiency, and 100-fold enhancement of the detection sensitivity was achieved compared with those obtained from normal capillary zone electrophoresis (CZE) or simple MEKC method. The proposed method was successfully applied for the determination of AAs in Chinese medicine preparations.     

Method validation for the determination of lead in raw cow's milk by electrothermal atomic absorption spectrometry

In this study, lead in raw cow's milk has been determined by validated electrothermal atomic absorption spectrometry (ETAAS) with Zeeman-effect background correction. Maximum pyrolysis and optimum atomization temperatures of lead were determined in the presence of modifiers. Pd + Mg(NO3)2 has been found a powerful modifier mixture for the determination of lead. The analytical parameters of the method such as limit of detection, limit of quantification and the effect of interfering ions have been investigated. The detection limit (3sigma) achieved by the method was calculated to be 0.62 ng/mL for Pb. Repeatability of the method evaluated as the relative standard deviation of 16-17 replicates using 5 ng/mL, under optimum experimental conditions were about 1.5% for synthetic sample solution and about 3% for real sample (N = 5). The described method has been validated by analyzing certified reference material (BCR-CRM 150) and by comparing the results with those obtained by inductively coupled plasma mass spectrometry (ICP-MS). The validated method was applied to raw cow's milk samples produced in 7 different regions of Turkey in 2003-2004. Raw cow's milk contained a mean (range) of 31.4 (2.5 - 313) microg kg(-1) lead with a relative error below 2%.     

Hydrophobic labeling of amino acids: transient trapping-capillary/microchip electrophoresis

Transient trapping (tr-trapping) was developed as one of the on-line sample preconcentration techniques to improve a low concentration-sensitivity in microchip electrophoresis (MCE), providing highly effective preconcentration and separation based on the trap-and-release mechanism. However, a poor performance to hydrophilic analytes limited the applicability of tr-trapping. To overcome this drawback, tr-trapping was combined with a sample labeling using a hydrophobic reagent in CE. Three commercially available fluorescent dyes, fluorescein isothiocyanate, succinimidyl esters of Alexa Fluor 488 and BODIPY FL-X, were tested as derivatization reagents to increase the hydrophobicity of amino acids (AAs) that were undetectable due to no fluorescence/UV-absorbance. As a result, it was confirmed that BODIPY labeling allowed various AAs to be analyzed in tr-trapping-micellar electrokinetic chromatography (tr-trapping-MEKC) by the increase in the hydrophobicity. In tr-trapping-MEKC, both the improvement of the resolution and 106-125-fold enhancements of the detectability of labeled AAs were achieved relative to the conventional capillary zone electrophoresis. The limit of detection of labeled phenylalanine was improved from 800 to 5 pM by applying tr-trapping-MEKC. In tr-trapping-microchip MEKC, furthermore, an 80-160-fold enhancement of the peak intensity and a baseline separation was also achieved within 30 s. These results clearly demonstrate that the tr-trapping technique with hydrophobic labeling will make CE/MCE more sensitive for various analytes.     

Determination of glyphosate and phosphate in water by ion chromatography--inductively coupled plasma mass spectrometry detection

Quantitative determination of trace glyphosate and phosphate in waters was achieved by coupling ion chromatography (IC) separation with inductively coupled plasma mass spectrometry (ICP-MS) detection. The separation of glyphosate and phosphate on a polymer anion-exchange column (Dionex IonPac AS16, 4.0 mm x 250 mm) was obtained by eluting them with 20 mM citric acid at 0.50 mL min(-1), and the analytes were detected directly and selectively by ICP-MS at m/z = 31. Parameters affecting their chromatographic behaviors and ICP-MS characteristics were systematically examined. Based on a 500-microL sample injection volume, the detection limits were 0.7 microgL(-1) for both glyphosate and phosphate, and the calibrations were linear up to 400 microgL(-1). Polyphosphates, aminomethylphosphonic acid (the major metabolite of glyphosate), non-polar and other polar phosphorus-containing pesticides showed different chromatographic behaviors from the analytes of interest and therefore did not interference. The determination was also interference free from the matrix anions (nitrate, nitrite, sulphate, chloride, etc.) and metallic ions. The analysis of certified reference material, drinking water, reservoir water and Newater yielded satisfactory results with spiked recoveries of 97.1-107.0% and relative standard deviations of < or = 7.4% (n = 3). Compared to other reported methods for glyphosate and phosphate, the developed IC-ICP-MS method is sensitive and simple, and does not require any chemical derivatization, sample preconcentration and mobile phase conductivity suppression.     

Determination of phenazopyridine in human plasma by GC-MS and its pharmacokinetics

A sensitive, selective, and simple gas chromatography-mass spectrometry method is developed for quantitation of phenazopyridine (PAP) in human plasma using internal standard (diazepam). PAP and IS are extracted from plasma by liquid-liquid extraction and analyzed on a DB-5MS column with mass selective detector. Excellent linearity is found between 5-500 ng/mL (r = 0.9992, n = 7) for PAP in human plasma. The limit of detection is 0.3 ng/mL. Intra- and Inter-day precisions expressed as the relative standard deviation for the method are 1.37-6.69% and 1.24-6.01%, respectively. Extraction efficiency is more than 90%, and recoveries are in the range of 92.65-96.21%. This method is successfully applied for the pharmacokinetics and bioequivalence of 2 formulations of PAP in 18 healthy male volunteers who received a single 200 mg dose of each formulation.     

Determination of trace metals by capillary electrophoresis

A new analytical procedure is developed using a strong complexing agent, 1,10-phenanthroline (Phen), for direct UV detection of Zn, Mn, Cu, Co, Cd, and Fe at microg/L concentrations in environmental water samples. The metal chelates formed showed different electrophoretic mobilities and solved the comigration problem for capillary electrophoresis (CE) separation of free metal ions. To obtain stable metal-Phen chelates during the capillary zone electrophoresis (CZE) run, both pre-column and on-column complexation are required and threefold excess of Phen over metal ions should be added to the sample. The optimized background electrolyte (BGE) consists of 30 mM hydroxylamine hydrochloride and 0.1% methanol at pH 3.6. Under hydrodynamic sampling, CE run at + 20 kV in 65 cm x 0.05 mm ID fused-silica column with detection at 265 nm, baseline separation, satisfactory working ranges (10 microg/L to 5.5 mg/L), sensitive detection limits (1-3 microg/L), good repeatability for migration times (relative standard deviation, RSD 0.36-0.81%, n = 5), peak area (RSD 3.2-4.2%, n = 5) and peak height (RSD 3.2-4.5%, n = 5) were obtained for the metal cations investigated. The reliability of the method was established by parallel determination using the inductively coupled plasma-atomic emission spectrometry (ICP-AES) method giving results within statistical variation. The procedure developed is shown to provide a quick, sensitive, precise, and economic method for simultaneous determination of metal cations that can form stable chelates with Phen.     

Assay of aromatic amino acid enantiomers in rice-brewed suspensions by chiral ligand-exchange CE

The aim of this work was to assay seasoning D- or L-aromatic amino acids (AAs) in rice-brewed suspensions, Laozao in Chinese, by chiral ligand-exchange CE with UV detection and Zn(II) complex as a chiral selecting system. Resolution and peak retention were found to be parallel to the basicity of the AA chiral ligands, and basic L-Arg was known to work the best at pH 8.20 compared with L-Lys and other AA ligands. Baseline separation of DL-aromatic AAs and partially separation of some FMOC-labeled nonaromatic AAs have been achieved using a running buffer of 5 mM ammonium acetate, 100 mM boric acid, 3 mM ZnSO(4), and 6 mM L-Arg at pH 8.20. The aromatic amino acids in four brands of Laozao were measured in a range of 0.25-20 microg/mL for Typ, 1.00-120 microg/mL for Phe, and 2.50-200 microg/mL for Tyr, with linear regression coefficient all over 0.999. The LOD (S/N=3) was 0.15 microg/mL for Typ, 0.50 microg/mL for Phe, and 1.25 microg/mL for Tyr. The recovery of the method determined by spiking with the supernates of Laozao as background was 94.0-112.9%. The RSDs of migration time and peak area measured from six injections of tyrosine were 0.2 and 2.7%, respectively, for run-to-run, and 1.6 and 3.2%, respectively for day-to-day. Interestingly, there were only L-Trp, D-Tyr, and L-Tyr found in the assayed four brands of Laozao. They may serve as an index to recognize the brand of Laozao.     

Qualitative analysis of some carboxylic acids by ion-exclusion chromatography with atmospheric pressure chemical ionization mass spectrometric detection

A simple, selective and sensitive method for the determination of carboxylic acids has been developed. A mixture of formic, acetic, propionic, valeric, isovaleric, isobutyric, and isocaproic acids has been separated on a polymethacrylate-based weak acidic cation-exchange resin (TSK gel OA pak-A) based on an ion-exclusion chromatographic mechanism with detection using UV-photodiode array, conductivity and atmospheric pressure chemical ionization mass spectrometry (APCI-MS). A mobile phase consisting of 0.85 mM benzoic acid in 10% aqueous methanol (pH 3.89) was used to separate the above carboxylic acids in about 40 min. For LC-MS, the APCI interface was used in the negative ionization mode. Linear plots of peak area versus concentration were obtained over the range 1-30 mM (r2=0.9982) and 1-30 mM (r2=0.9958) for conductimetric and MS detection, respectively. The detection limits of the target carboxylic acids calculated at S/N=3 ranged from 0.078 to 2.3 microM for conductimetric and photometric detection and from 0.66 to 3.82 microM for ion-exclusion chromatography-APCI-MS. The reproducibility of retention times was 0.12-0.16% relative standard deviation for ion-exclusion chromatography and 1.21-2.5% for ion-exclusion chromatography-APCI-MS. The method was applied to the determination of carboxylic acids in red wine, white wine, apple vinegar, and Japanese rice wine.     

Continuous solid-phase extraction method for the determination of amines in human urine following on-line microwave-assisted acid hydrolysis

Humans are exposed to endogenous or exogenous formation of aromatic amines (AAs) and N-nitrosamines (NAms), which are considered to be potent carcinogens. The objective of this study was to monitor AAs and NAms in human urine to obtain a way to assess exposure. However, while NAms can be directly detected in urine samples, AAs require hydrolysis to convert their conjugates into free amines. A semiautomatic flow-base method is proposed for the simultaneous determination of aliphatic and aromatic NAms, anilines and chloroanilines in human urine in one analytical run. Conjugated AAs are released from urine by on-line microwave-assisted acid hydrolysis without degradation of NAms; all amines were then preconcentrated using solid-phase extraction. Separation/determinations are carried out by gas chromatography and mass spectrometry operating in selected ion monitoring mode. The method is fast (∼15 min for 25 mL of sample) and provides low limits of detection (from 2 to 26 ng/L) with good precision (relative standard deviation within and between days less than 7%). Finally, the proposed method was successfully applied to check AAs and NAms in the human urine of exposed and unexposed researchers. The kinetics of amine excretion in the urine of the researchers exposed is calculated after termination of the exposure and shows half-life times between 1.3 and 2.1 h, and that the dosage absorbed was eliminated within 6 h after exposure.     

Liquid chromatography with electrospray ionization mass spectrometry method for the assay of glucosamine sulfate in human plasma: validation and application to a pharmacokinetic study

A liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method was developed and validated for the assay of glucosamine sulfate in human plasma. Plasma proteins were precipitated by acetonitrile, followed by vortex mixing and centrifugation. The supernatant was transferred and derivatized with phenyl iso-thiocyanate in acetonitrile at 60 degrees C for 40 min. Chromatographic separation was performed on a C(18) column (Inertsil ODS-3 150 x 2.1 mm i.d., 5 microm, JP) with a mobile phase gradient consisting of 0.2% acetic acid (aqueous) and methanol at a flow-rate of 0.3 mL/min. MS detection using electrospray ionization (ESI) as an interface was used in single ion monitoring mode to determine positive ions at m/z 297. This method was shown to be selective and sensitive for glucosamine sulfate. The limit of detection was 35 ng/mL for glucosamine sulfate in plasma and the linear range was 0.1-20 microg/mL in plasma with a correlation coefficient (r) of 0.9991. The relative standard deviations (RSDs) of intra-day and inter-day assays were 8.7-11.4 and 9.8-12.6%, respectively. Extraction recoveries of glucosamine sulfate in plasma were greater than 73%. This method proved to be simple, reproducible and feasible for pharmacokinetic studies of glucosamine sulfate in healthy volunteers after a single oral administration (1500 mg). The pharmacokinetic parameters and relative bioavailabilities were investigated for both domestic glucosamine sulfate tablet and capsule preparations compared with an imported capsule product.     

离子转换色谱-紫外检测法测定啤酒中的无机阴离子和有机酸

建立了离子转换色谱与紫外检测器联用检测啤酒中无机阴离子与有机酸的新方法。在传统的离子色谱基础上引入两根离子转换柱,无机阴离子与有机酸经过两步转换,定量转换成有相同紫外响应的碘酸盐,然后用紫外检测器代替电导检测器进行定量分析。在Dionex AS11-HC阴离子色谱柱上,采用KOH梯度淋洗方式实现了12种无机阴离子与有机酸的分离。结果表明,12种无机阴离子和有机酸检出限（S/N=3）与定量限（S/N=10）分别为6.168~29.01 μg/L与20.56~66.30 μg/L;线性关系良好（r在0.9994以上）,回收率为89.0%~117.0%,RSD均小于1.0%,该方法与传统的电导检测器检测结果相当。该方法简单快捷,只需采用少数标准曲线,就可以实现啤酒中无机离子与有机酸的定量测定。