Peak Area Ratios Of Analytes Research Articles

Quantitation of Mycophenolic Acid and Mycophenolic Acid Glucuronide in Serum or Plasma by LC-MS/MS.

Mycophenolic acid (MPA) is an immunosuppressant that is used as an adjunct therapy in renal, liver, and heart transplantation. Due to its narrow therapeutic range, monitoring MPA levels is essential to avoid toxicity and organ rejection. Although immunoassays are available for the determination of MPA, mass spectrometry methods are preferred due to their higher specificity. Herein, we describe a liquid chromatography tandem mass spectrometry (LC-MS/MS) method utilizing positive ionization electrospray and multiple reaction monitoring (MRM) for the quantification of MPA levels and its conjugate, MPA glucuronide (MPAG). Blood collected in a plain, EDTA, or heparin-containing tube is centrifuged to separate the serum or plasma. Proteins are precipitated using a zinc sulfate solution and acetonitrile containing deuterated internal standards (MPA-d3 and MPAG-d3). The resulting protein-free supernatant is injected into the LC-MS/MS system for analysis. The chromatography involves the use of a C18 column and ammonium acetate/water/formic acid and ammonium acetate/methanol/formic acid mobile phases. Quantification of MPA and MPAG levels is achieved by comparing the MRM peak area ratios of analytes and internal standards, consisting of specific precursor/product pairs, with those of calibrators at various concentrations. Calibration curves are constructed from the MRM peak area ratios of calibrators and internal standards versus concentration. © 2023 Wiley Periodicals LLC. Basic Protocol: Quantitation of mycophenolic acid and mycophenolic acid glucuronide in serum or plasma by LC-MS/MS.

Technical note: simultaneous determination of amino thiols in pig tissue by ultra-high performance liquid chromatography with fluorescence detection.

Sulfur amino acid nutrition and metabolism are linked to animal disease. While validated methods for the determination of amino thiol levels in plasma or serum are available, there is a dearth of validated methods for their measurement in tissue. A robust and reproducible ultra-high performance liquid chromatography method has been validated for the simultaneous determination of concentrations of cysteine (Cys), cysteinylglycine (CysGly), homocysteine (Hcys), γ-glutamylcysteine (γ-GluCys), and glutathione (GSH) in pig tissue. Tissue was homogenized and deproteinized with trichloroacetic acid. Amino thiols in the acid-soluble fraction of the tissue homogenate were reduced with tris-(2-carboxyethyl)-phosphine hydrochloride and derivatized with 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F). Amino thiols were resolved under reversed-phase gradient conditions on a Waters Acquity BEH C18 column (1.7 µm, 2.1 mm × 100 mm) within 4.5 min and detected with fluorescence. The peak area ratio of analyte to 2-mercaptopropionylglycine internal standard, added to external calibration standards and samples, was used to develop linear calibration curves. Linear calibrations were performed over the range of 15-1,500 nmol/g for Cys, CysGly, Hcys, and γ-GluCys and 150-15,000 nmol/g for GSH. Linearity, lower limit of detection, lower limit of quantitation, accuracy, precision, sample stability, and carryover were evaluated. We demonstrate excellent linearity for all analytes within their respective concentration range (r2 > 0.99) and excellent recovery of amino thiols from spiked samples (mean ± SD across tissues; Cys, 100.0 ± 2.2%; CysGly, 95.4 ± 5.1%; Hcys, 96.6 ± 2.0%; γ-GluCys, 102.2 ± 2.7%; and GSH, 100.6 ± 3.3%). The intra-day and inter-day precisions did not exceed 5% and 10%, respectively. Repeated freezing and thawing of tissue homogenate did not affect measured amino thiol concentrations, ABD-labeled amino thiols were stable for 1 wk after derivatization, and there was no sample carryover across consecutive injections. We confirm the identity of each ABD-labeled amino thiol with Orbitrap mass spectrometry. Finally, we apply the method to the determination of amino thiol concentrations in liver and jejunum tissues in newly weaned pigs and show that despite elevated Cys and maintained GSH concentrations in liver, both γ-GluCys and GSH decline in jejunum of weaned pigs.

Capillary electrophoresis-UV analysis using silica-layer coated capillary for separation of seven phenolic acids and caffeine and its application to tea analysis

This work presents an innovative silica-layer coated capillary with comparison study of the silica-layer coated capillary and the fused-silica capillary for the separation of seven phenolic acids viz. p-hydroxyphenylacetic acid (PHPA), p-coumaric acid (PCA), p-hydroxybenzoic acid (PHBA), caffeic acid (CFA), (3,4-dihydroxyphenyl)acetic acid (DHPA), gallic acid (GLA), and 2,3,4-trihydroxybenzoic acid (THBA), together with caffeine (CF), by capillary electro-chromatography (CEC) and micellar electrokinetic chromatography (MEKC), respectively. The running buffer was 25.0 mM borate at pH 9.0, with addition of 50.0 mM sodium dodecyl sulfate for the MEKC mode. The non-coated capillary could not separate all seven phenolic acids by CEC or MEKC. This was achieved using the coated capillary for both CEC and MEKC. The innovative coated capillary with CEC had plate number N ≥ 2.0 × 104 m−1 and resolution Rs ≥ 1.6 for all adjacent pairs of peaks. The capillary was also able to separate GLA and THBA which are structural isomers. Although MEKC mode provided comparable efficiency and selectivity, the reduced EOF of the coated capillary led to longer separation time. The linear calibration range of the seven phenolic acids and caffeine were different but the coefficients of determinations (r2) were all > 0.9965. The precisions of the relative migration times and peak area ratios of analyte to internal standard were 0.1–1.8% and 1.8–6.8%, respectively. There were no statistical differences in the efficiency of separation of the phenolic acids and caffeine for three coated capillaries. It was applied to the analysis of caffeine and phenolic acids in brewed tea using tyramine as the internal standard. The tea samples were diluted prior to analysis by CEC. The separation was less than 15 min. Caffeine, gallic acid and p-coumaric acid were detected and quantified. Caffeine and gallic acid contents were 10.8–15.0 and 2.6–4.8 mg g−1 dry tea leaves, respectively. p-Coumaric acid was detected in only one of the samples with a content of 0.4 mg g−1. Percent recoveries of spiked diluted samples were 90 ± 9 to 106 ± 13%, respectively.Article highlightsSilica-layer coated capillary is first reported for simultaneous separation of seven phenolic acids by non-MEKC analysis.Performance between coated, and non-coated capillaries with analysis by CEC and MEKC were compared.Plate number, resolution, capillary reproducibility, and electroosmotic flow mobility are investigated.Graphical abstract

Determination of Mycophenolic Acid and Mycophenolic Acid Glucuronide Using Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS).

Mycophenolic acid (MPA) is an immunosuppressant that is used in renal, liver, and heart transplantation. Due to its narrow therapeutic range, monitoring of MPA levels is essential to avoid toxicity and organ rejection. Although immunoassays are available for the determination of MPA, due to their higher specificity, mass spectrometry methods are preferred. In this unit, we describe a liquid chromatography tandem mass spectrometry (LC/MS/MS) method utilizing positive ionization electrospray and multiple reaction monitoring (MRM) for the quantification of levels of MPA and its conjugate MPA glucuronide (MPAG). Blood collected in a plain, EDTA, or heparin-containing tube is centrifuged to separate the serum or plasma. Proteins are precipitated using a solution containing zinc sulfate and acetonitrile that has been spiked with deuterated internal standards. The resulting protein-free supernatant is injected into the LC/MS/MS system for analysis. The chromatography involves the use of a C18 column and ammonium acetate/water/formic acid and ammonium acetate/methanol/formic acid mobile phases. Quantification of MPA and MPAG levels is achieved by comparing the MRM peak area ratios of analytes and internal standards, consisting of specific precursor/product pairs, with those of calibrators at various concentrations. Calibration curves are constructed from the MRM peak area ratios of calibrators and internal standards versus concentration. © 2018 by John Wiley & Sons, Inc.

Separation and determination of chlorophenylpiperazine isomers in confiscated pills by capillary electrophoresis

A simple capillary electrophoretic method with spectrophotometric UV detection at 236nm has been developed for the selective separation and determination of 1-(2-chlorophenyl)piperazine (oCPP), 1-(3-chlorophenyl)piperazine (mCPP) and 1-(4-chlorophenyl)piperazine (pCPP) in confiscated pills. Several cyclodextrin derivatives were tested to compose the background electrolyte (BGE). The optimized BGE contained 20mmol/L phosphoric acid adjusted to pH 2.5 with triethylamine and 10mmol/L α-cyclodextrin, which provided acceptable resolution of analytes and candidate interferents in less than 15min. The analyses were performed at constant voltage of 25kV in 60cm (effective length 50cm; 50μm i.d.) uncoated fused-silica capillary maintained at 25°C with sample injection at 4826Pa for 8s. Procaine at a concentration of 0.1mg/mL was used as internal standard (IS). Possible interference from other drugs such as amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxy-N-ethylamphetamine, 1-(3-trifluoromethylphenyl)piperazine and cocaine was also examined. The analytical curves were linear (R2=0.9994–0.9995) in the range of 10–200μg/mL (for oCPP and mCPP) and 20–200μg/mL for pCPP. Limits of detection (LODs) were 2.0μg/mL (oCPP), 2.5μg/mL (mCPP) and 3.5μg/mL (pCPP). Intraday precision at three concentration levels and six replicates of each level (10, 100, 200μg/mL of each analyte; n=18) was evaluated for the corrected peak area ratio of analyte to IS and the migration times giving RSDs≤4.9%. The accuracy was estimated for mCPP by a recovery test at the same three concentration levels and recoveries varied from 101.0 to 101.6%. The method has been successively applied to the analysis of 17 confiscated pills based mostly on mCPP.

Simultaneous Estimation of Fluoxetine and Norfluoxetine in Plasma by RP-HPLC Employing Pre-Column Derivatization for UV-Sensitivity Enhancement

A rapid high-performance liquid chromatographic method is described for the simultaneous estimation of the widely used antidepressant drug, fluoxetine and its principal metabolite norfluoxetine in human plasma. Pre-column derivatization of norfluoxetine was done using 4-dimethylaminobenzaldehyde (PDAB) to overcome the limitations of sensitivity of norfluoxetine. After liquid-liquid extraction the separation of analytes and internal standard from endogenous matrix interference was achieved using a reversed-phase HIQ sil ODS column (250 mm length x 4.6 mm internal diameter) KYA TECH (Japan) and assayed by ultraviolet absorption at 227 nm. The isocratic mobile phase (1 ml/min.) consisting of acetonitrile: water: triethylamine: 0.01 M Orthophospheric acid (O.P.A.) (70:30:0.5:2) was used to separate fluoxetine, norfluoxetine and the internal standard, nebivolol. The relative retention times were 2.49, 4.24 and 7.29 min for norfluoxetine, fluoxetine and nebivolol, respectively. Chromatographic run time was 10 min and peak area ratios of analytes to IS were used for regression analysis of calibration curve. Linearity was obtained over the concentration range 10-60 μg/ml for both substances. The mean % recovery ± SD was found to be 101.23% ± 1.0 and 100.69 ± 0.67 for fluoxetine and norfluoxetine respectively. The method seems suitable, in terms of accuracy and precision, for the determination of fluoxetine plasma levels of patient; furthermore, it is rapid and sensitive.

Effect of nicotine on cytochrome P450 1A2 activity

Effect of nicotine on cytochrome P450 1A2 activity

Simultaneous quantitative analysis of bioactive sphingolipids by high-performance liquid chromatography-tandem mass spectrometry

There has been a recent explosion in research concerning novel bioactive sphingolipids (SPLs) such as ceramide (Cer), sphingosine (Sph) and sphingosine 1-phosphate (Sph-1P) that necessitates development of accurate and user-friendly methodology for analyzing and quantitating the endogenous levels of these molecules. ESI/MS/MS methodology provides a universal tool used for detecting and monitoring changes in SPL levels and composition from biological materials. Simultaneous ESI/MS/MS analysis of sphingoid bases (SBs), sphingoid base 1-phosphates (SB-1Ps), Cers and sphingomyelins (SMs) is performed on a Thermo Finnigan TSQ 7000 triple quadrupole mass spectrometer operating in a multiple reaction monitoring (MRM) positive ionization mode. Biological materials (cells, tissues or physiological fluids) are fortified with internal standards (ISs), extracted into a one-phase neutral organic solvent system, and analyzed by a Surveyor/TSQ 7000 LC/MS system. Qualitative analysis of SPLs is performed by a Parent Ion scan of a common fragment ion characteristic for a particular class of SPLs. Quantitative analysis is based on calibration curves generated by spiking an artificial matrix with known amounts of target synthetic standards and an equal amount of IS. The calibration curves are constructed by plotting the peak area ratios of analyte to the respective IS against concentration using a linear regression model. This robust analytical procedure can determine the composition of endogenous sphingolipids (ESPLs) in varied biological materials and achieve a detection limit at 1pmol or lower level. This and related methodology are already defining unexpected specialization and specificity in the metabolism and function of distinct subspecies of individual bioactive SPLs.

Determination of Sophocarpine, Matrine, and Sophoridine in KUHUANG Injection by GC-MS

A GC-MS procedure was developed to determine sophocarpine (SC), matrine (MT), and sophoridine (SRI) in KUHUANG injection. The chromatographic separation was performed on an HP-5MS column (30 m × 0.35 mm inner diameter, 0.25 µm film thickness) with helium as the carrier gas. The oven temperature was programmed from 170 to 205°C at a rate of 0.8 K/min, and n-tetracosane was used as the internal standard (IS). There was a good linear relationship between the peak area ratio of analyte to IS and the concentration of analyte in the ranges 0.0360–0.100 mg/mL for SC, 0.0461–0.346 mg/mL for MT, and 0.0473–0.355 mg/mL for SRI. The recoveries were 86.1–100.9% for SC, 87.9–104.0% for MT, and 92.5–107.2% for SRI. The interday and intraday relative standard deviations (RSDs) of determinations were less than 4.6, 2.7, and 5.1% for the assay of SC, MT, and SRI, respectively. The limits of detection (LOD) were 0.018 mg/mL for SC, 0.0231 mg/mL for MT, and 0.0287 mg/mL for SRI. The limits of quantitation (LOQ) were 0.036 mg/mL for SC, 0.0461 mg/mL for MT, and 0.0473 mg/mL for SRI. The results indicate that the developed method can be used to improve the quality control of KUHUANG injection.

Rapid quantitation of fluoxetine and norfluoxetine in serum by micro-disc solid-phase extraction with high-performance liquid chromatography–ultraviolet absorbance detection

A rapid, robust and sensitive method for the extraction and quantitative analysis of serum fluoxetine (FLX) and norfluoxetine (N-FLX) using a solid-phase extraction (SPE) column and high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection was developed and validated. The sample clean-up step was performed by simple micro-disc mixed-mode (non-polar and strong cation exchange (SCX)) SPE cartridges. Separation of analytes and internal standard (IS) clomipramine (CLO) from endogenous matrix interference was achieved using a Waters Symmetry C 8 ( 150 mm×2.1 mm i.d., 5 μm) reversed-phase narrow bore column. The relative retention times were 8.5, 9.6 and 10.5 min for FLX, N-FLX and CLO, respectively with a low isocratic flow rate of 0.3 ml/min. Chromatographic run time was completed in 15 min and peak area ratios of analytes to IS were used for regression analysis of the calibration curve. The latter was linear from 10 to 4000 nmol/l using 0.5 ml sample volume of serum. The average recovery was 95.5% for FLX and 96.9% for N-FLX. The lowest limit of quantitation (LLOQ) for serum FLX and N-FLX was 10 nmol/l (on-column amount of 200 fmol). The method described was used to analyse serum samples obtained from rats given chronic FLX treatment and to examine the relationship between steady state serum drug concentrations and neurochemical changes in several brain regions.

Determination of dextromethorphan and its metabolite dextrorphan in human urine by capillary gas chromatography without derivatization

A sensitive, simple and accurate method was developed for determination of dextromethorphan (DM) and dextrorphan (DT) in human urine by capillary gas chromatography without derivatization. After an oral dose of 30 mg DM, urine samples were collected and extracted, then analyzed on 0.22 mm×17 m HP-1 capillary column. DM and its metabolite DT were analyzed simultaneously with good separation. Docosane was used as the internal standard (I.S.). The detector used was flame ionization detector (FID). There was a linear relationship between peak area ratios of analytes to I.S. and concentration of analytes over the concentration range 0.37–7.38 μmol/l for DM and 0.39–77.8 μmol/l for DT. The recovery was 88.1∼103.9% for DM and 86.7∼96.8% for DT. The within-day and between-day coefficients of variation were less than 7.4 and 7.3% (RSD) for the assay of DM and DT in urine, respectively. The limits of detection (LOD) were 0.30 μmol/l for DM and 0.16 μmol/l for DT. The limits of quantitation (LOQ) were 0.37 μmol/l (RSD<6%) for DM and 0.39 μmol/l (RSD<7%) for DT. The method has been applied to determine the oxidative phenotypes of cytochrome P450 2D6 ( CYP2D6) in a Chinese population with metabolic ratio of DM in human urine.