A sensitive and specific liquid chromatography/tandem mass spectrometry method for determination of pinaverium bromide in human plasma: application to a pharmacokinetic study in healthy volunteers

A sensitive and specific method using liquid chromatography–electrospray tandem mass spectrometry (LC‐MS/MS) for the determination of pinaverium bromide in human plasma was developed and validated. Pinaverium bromide and an internal standard (paclitaxel) were isolated from plasma samples by precipitating plasma, and determined by LC‐MS/MS in multiple‐reaction monitoring mode. The main metabolite of pinaverium bromide and endogenous substances in plasma did not show any interference. The calibration curve was linear over the plasma concentration range of 10.0–10000.0 pg/mL with a correlation coefficient of 0.9979. The relative standard derivations intra‐ and inter‐day at 30.0, 300.0 and 8000.0 pg/mL in plasma were less than 15%. The absolute recoveries of pinaverium bromide and the internal standard were 99.7–111.7 and 106.2%, respectively. The lower limit of quantitation was 10 pg/mL. The analytical method was successfully applied to study the pharmacokinetics of pinaverium bromide tablets in healthy Chinese volunteers. Copyright © 2011 John Wiley & Sons, Ltd.     

Sensitive and rapid liquid chromatography/tandem mass spectrometry assay for the quantification of amlodipine in human plasma

A simple, sensitive and rapid high-performance liquid chromatography/electrospray ionization tandem mass spectrometry method was developed and validated for the assay of amlodipine in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse-phase C(18) column and analyzed by MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 409/238 for amlodipine and m/z 409/228 for the IS. The assay exhibited a linear dynamic range of 50-10,000 pg/mL for amlodipine in human plasma. The lower limit of quantification was 50 pg/mL with a relative standard deviation of less than 8%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The average absolute recoveries of amlodipine and the IS from spiked plasma samples were 74.7 +/- 4.6 and 72.1 +/- 2.0%, respectively. A run time of 1.5 min for each sample made it possible to analyze more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies. The observed maximum plasma concentration (Cmax) of amlodipine (2.5 mg oral dose) was 1425 pg/mL, time to observed maximum plasma concentration (Tmax) was 8.1 h and elimination half-life (T(1/2)) was 50.1 h.     

Fast and simple online sample preparation coupled with capillary LC-MS/MS for determination of prostaglandins in cell culture supernatants

An online 2-D strong cation exchange (SCX)-RP capillary liquid chromatographic (cLC) method with IT mass spectrometric (IT-MS/MS) detection for the simultaneous determination of prostaglandin (PG) A(1), PGD(2), PGE(1), PGE(2), PGF(2a), 6-keto-(6k)PGF(1a), and 15-Delta(12,14)-deoxy-PGJ(2) (15dPGJ(2)) in cell culture supernatants was developed and validated. Pretreatment of the cell culture supernatants included dilution and filtration, and the analysis time including all sample preparation steps was less than 50 min per sample. Peptides/proteins contained in the matrix were removed by the SCX column. LODs in the range of 0.4-2.2 ng/mL cell culture supernatant, recoveries higher than 80% and within- and between-day precisions of less than 30% RSDs were obtained. Human mesenchymal stem cells (hMSCs) were stimulated with cytokine-containing supernatants derived from activated T lymphocytes, and PG production was analyzed using the developed method. PGE(2 )was found in cultures from both untreated and stimulated hMSCs, while PGE(1) was present above the detection limit only in stimulated cells.     

Development and validation of an LC-MS/MS method for profiling 39 urinary steroids (estrogens,androgens, corticoids,and progestins)

Abnormal production or metabolism of steroid hormones is responsible for the development of endocrine diseases. Thus, accurate quantification of steroid hormones is needed for both research into clinical conditions and diagnostic and monitoring purposes. An improved analytical method for profiling 39 steroids in urine using LC–MS/MS was developed. As a pre-treatment procedure prior to LC–tandem mass spectrometry (LC–MS/MS) analysis, hydrolysis using β-glucuronidase and solid-phase extraction for purifying the samples were performed. Steroids were separated using Waters ACQUITY BEH C₁₈ column (2.1 × 100 mm, 1.7 μm) and a mobile phase consisting of eluent A (0.01% formic acid and 1 mm ammonium formate in water) and eluent B (0.01% formic acid and 1 mm ammonium formate in methanol) with a gradient program at a flow rate of 0.4 mL/min. Under the optimized method, the linearity of calibration curves was higher than 0.992. The limits of detection at signal-to-noise ratio of 3 were 0.03–90 ng/mL. The developed novel LC–MS/MS method can quantitatively profile 39 steroids in a single analytical run. Steroid profiling based on quantitative results could improve the diagnosis and monitoring of hormone-dependent diseases.     

Selective clean-up method using an immunoaffinity column following radioimmunoassay of prostaglandin F2 alpha in biological fluids.

A selective clean-up method using an immunoaffinity column followed by radioimmunoassay (RIA) was developed for determining prostaglandin F2 alpha (PGF2 alpha) in human urine and plasma. Polyclonal antibody raised against PGF2 alpha, obtained from rabbits, was coupled to a tresyl-activated support based on a synthetic hydrophilic resin, TSKgel Tresyl-Toyopearl 650M, and used as the stationary phase for the immunoaffinity column. A human urine or plasma sample was introduced to this column, and PGF2 alpha was eluted with methanol-water (50:50, v/v) after the column had been washed. The eluate was subjected to competitive RIA for PGF2 alpha. The cross-reactivities of the RIA to a number of endogenous prostanoids, except PGD2, were negligible and the sensitivity was 4 pg/tube (p less than 0.05), giving a detection limit of 40 pg/ml when 1 ml of plasma or urine was available. The recoveries of plasma and urine samples were 98-108% and 96-106%, respectively, and their assay variances were 7-23%. The concentrations of endogenous PGF2 alpha in plasma and urine used here were estimated to be 72 and 98 pg/ml, respectively. This method should be very useful for various biological samples because of its good specificity, sensitivity, reliability and reproducibility.     

Determination of clopidogrel in human plasma by liquid chromatography/tandem mass spectrometry: application to a clinical pharmacokinetic study

A rapid and sensitive LC/MS/MS assay was developed and validated for the determination of clopidogrel in human plasma. Clopidogrel was extracted by single liquid-liquid extraction with pentane, and chromatographic separations were achieved on a C(18) column. The method was validated to demonstrate the specificity, linearity, recovery, lower limit of quantification (LLOQ), stability, accuracy and precision. The multiple reaction monitoring was based on m/z transition of 322.2 --> 211.9 for clopidogrel and 264.1 --> 125.1 for ticlopidine (internal standard). The total analytical run time was relatively short (3 min), and the LLOQ was 10 pg/mL using 0.5 mL of human plasma. The assay was linear over a concentration range from 10 to 10,000 pg/mL (r > 0.999). The intra- and inter-day accuracies were 101.3-108.8 and 98.4-103.5%, respectively, and the intra- and inter-day assay precisions were 1.9-5.5 and 4.4-8.1%, respectively. The developed assay method was applied to a pharmacokinetic study in human volunteers after oral administration of clopidogrel at a dose of 150 mg.     

Development and application of a new on-line SPE system combined with LC-MS/MS detection for high throughput direct analysis of pharmaceutical compounds in plasma

A technique using a fully automated on-line solid phase extraction (SPE) system (Symbiosis, Spark Holland) combined with liquid chromatography (LC)-mass spectrometry (MS/MS) has been investigated for fast bioanalytical method development, method validation and sample analysis using both conventional C18 and monolithic columns. Online SPE LC-MS/MS methods were developed in the automated mode for the quantification of model compounds (propranolol and diclofenac) directly in rat plasma. Accuracy and precision using online SPE LC-MS/MS with conventional C18 and monolithic columns were in the range of 88-111% and 0.5-14%, respectively. Total analysis cycle time of 4 min per sample was demonstrated using the C18 column. Monolithic column allowed for 2 min total cycle time without compromising the quality and validation criteria of the method. Direct plasma sample injection without on-line SPE resulted in poor accuracy and precision in the range of 41-108% and 3-81%. Furthermore, the increase in back pressure resulted in column damage after the injection of only 60 samples.     

Liquid chromatography-tandem mass spectrometry for the determination of a new oxazolidinone antibiotic DA-7867 in human plasma

A liquid chromatography-tandem mass spectrometric (LC/MS/MS) method for the determination of a new ox-azolidinone antibiotic DA-7867, (S)-[N-3-(4-(2-(1-methyl-5-tetrazolyl)-pyridine-5-yl)-3- fl uorophenyl)-2-oxo-5-oxazolidinyl]methyl acetamide, in human plasma was developed. DA-7867 and internal standard, linezolid, were extracted from human plasma with ethyl acetate at acidic pH. A reverse-phase LC separation was performed on Luna C(8) column with the mixture of acetonitrile-ammonium formate (10 mm, pH 4.5; 35:65, v/v) as mobile phase. The analytes were determined using an electrospray ionization tandem mass spectrometry in the multiple-reaction-monitoring mode. The lower limits of quanti fi cation for DA-7867 was 2.5 ng/mL. The single liquid-liquid extraction quantitatively recovered DA-7867 and internal standard from plasma samples at the ranges of 82.2-86.7%. DA-7867 was stable in blank human plasma at room temperature for 24 h and following three freeze-thaw cycles.     

Determination of telmisartan in human blood plasma: Part II: Liquid chromatography-tandem mass spectrometry method development, comparison to immunoassay and pharmacokinetic study

A new liquid chromatography/atmospheric pressure chemical ionization-tandem mass spectrometry (LC/APCI-MS/MS) method with on-line sample clean-up for the determination of telmisartan in human blood plasma is presented. This technique is compared to a previously introduced enzyme-linked immunosorbent assay (ELISA), where fluorescence is used as detection method. For the LC/MS method applying an internal calibration via a deuterated internal standard, the limit of detection was 0.3 ng/mL, the limit of quantification was 0.9 ng/mL and the linear range extended from 0.9 to 1000 ng/mL. Forty-eight plasma samples from four healthy volunteers were analyzed in a pharmacokinetic study to obtain data for the method comparison. As a result, these two new and independent analytical methods for the determination of telmisartan in human blood plasma proved to yield comparable results for the amount of analyte.     

Determination of Tetracyclines in Honey Using Liquid Chromatography with Ultraviolet Absorbance Detection and Residue Confirmation by Mass Spectrometry

A determination method has been optimized and validated for the simultaneous analysis of tetracycline （TC）, oxytetracycline （OTC）, chlortetracycline （CTC） and doxycycline （DC） in honey. Tetracyclines （TCs） were removed from honey samples by chelation with metal ions bound to small Chelating Sepharose Fast Flow columns and eluted with Na2EDTA-Mcllvaine pH 4.0 buffers. Extracts were further cleaned up by Oasis HLB solid-phase extraction （SPE）, while other solid-phase extraction cartridges were compared. Chromatographic separation was achieved using a polar end-capped C 18 column with an isocratic mobile phase consisting of oxalic acid, acetonitrile and methanol. LC with ultraviolet absorbance at 355 nm resulted in the quantitation of all four tetracycline residues from honey samples fortified at 15, 50, and 100 ng/g, with liner ranges for tetracyclines of 0.05 to 2 μg/mL. Mean recoveries for tetracyclines were greater than 50% with R.S.D. values less than 10% （n= 18）. Detection limits of 5, 5, 10, 10 ng/g for oxytetracycline, tetracycline, chlortetracycline and doxycycline, respectively and quantitation limits of 15 ng/g for all the four tetracyclines were determined. Direct confirmation of the four residues in honey （2-50 ng/g） was realized by liquid chromatography-tandem mass spectrometry （LC/MS/MS）. The linear ranges of tetracyclines determined by LC/MS/MS were between 5 to 300 ng/mL, with the linear correlation coefficient r〉 0.995. The limits of detection of 1 to 2 ng/g were obtained for the analysis of the TCs in honey.     

Detecting pM concentrations of prostaglandins in cell culture supernatants by capillary SCX-LC-MS/MS

A highly sensitive, improved online strong cation exchange (SCX)--RP capillary liquid chromatographic (cLC) method with IT mass spectrometric (IT-MS/MS) detection for the simultaneous determination of prostaglandin (PG)A(1), PGD(2), PGE(1), PGE(2), PGF(2alpha), 8-iso-(8i)PGF(2alpha), 6-keto-(6k)PGF(1alpha), and 15-Delta(12, 14)-deoxy-PGJ(2) (15dPGJ(2)) in cell culture supernatants was developed and validated. Pretreatment of the cell culture supernatants included only dilution and filtration, and the analysis time including all sample preparation steps was 60 min per sample. Peptides/proteins contained in the matrix were removed by the SCX column. LODs in the range of 8-44 pg/mL (25-120 pM) cell culture supernatant were obtained. Excellent linearity (R(2) > 0.99) and satisfactory recoveries and within- and between-day precisions were obtained. Human mesenchymal stem cells (hMSCs) were stimulated with tumor necrosis factor alpha (TNFalpha) or TNFalpha/IL-17, and PG production was analyzed using the developed method. The four PGs, 6kPGF(1a), PGF(1a), PGE(2), and PGE(1 )were detected both in nonstimulated and stimulated cells. The amount of PG produced by the cell increased when the cell was stimulated.     

Quantification of polygalasaponin F in rat plasma using liquid chromatography–tandem mass spectrometry and its pharmacokinetics application

A rapid and highly selective liquid chromatography–tandem mass spectrometric (LC‐MS/MS) method for determination of polygalasaponin F (PF) in rat plasma was developed and validated. The chromatographic separation was achieved on a reverse‐phase Zorbax SB‐C₁₈ column (150 × 4.6 mm, 5 µm), using 2 mm ammonium acetate (pH adjusted to 6.0 with acetic acid) and acetonitrile (25:75, v/v) as a mobile phase at 30 °C. MS/MS detection was performed using an electrospray ionization operating in positive ion multiple reaction monitoring mode by monitoring the ion transitions from m/z 1091.5 → 471.2 (PF) and m/z 700.4 → 235.4 (internal standard), respectively. The calibration curve showed a good linearity in the concentration range 0.0544–13.6 µg/mL, with a limit of quantification of 0.0544 µg/mL. The intra‐ and inter‐day precisions were <9.7% in rat plasma. The method was validated as per US Food and Drug Administration guidelines and successfully applied to pharmacokinetic study of PF in rats. Copyright © 2015 John Wiley & Sons, Ltd.     

High‐performance liquid chromatography tandem mass spectrometry method for the determination of 2CC‐NBOMe and 25I‐NBOMe in human serum

2CC‐NBOMe {4‐chloro‐2,5‐dimethoxyphenethyl‐N‐[(2‐methoxyphenyl) methyl] ethanamine} and 25I‐NBOMe {2‐(4‐iodo‐2,5‐dimethoxyphenyl)‐N‐[(2‐methoxyphenyl) methyl] ethanamine} are of a class of N‐benzyl phenethylamine derivatives whose synthesis was first reported in the scientific literature in 2011. Recent reports from ‘personal drug experience websites’ and in the popular press indicate these drugs are the latest in a series of designer ‘Bath Salt’ drugs of abuse. The presented high‐performance liquid chromatography triple quadrupole mass spectrometry (HPLC/MS/MS) method was developed for the detection and quantification of 2CC‐NBOMe and 25I‐NBOMe in serum of intoxicated emergency department patients. The assay applies 2‐?(2,?5‐?dimethoxyphenyl)‐?N‐?(2‐?methoxybenzyl) ethanamine (25H‐NBOMe) as the internal standard. Samples were extracted using solid‐phase extraction columns. The chromatographic separation was performed on a Luna 3 µ C₈(2) 100 Å, 100 × 2.0 mm, column. Detection was accomplished by multiple‐reaction monitoring via an electrospray ionization source operating in the positive ionization mode. The calibration curves were linear over the investigated concentration range, 30–2000 pg/mL, with a lower limit of detection of 10 pg/mL for both 2CC‐NBOMe and 25I‐NBOMe. The method proved suitable for serum clinical toxicology testing. Two severely intoxicated emergency department patients were determined to have serum concentrations of 250 and 2780 pg/mL of 25I‐NBOMe using the presented method. Copyright © 2013 John Wiley & Sons, Ltd.     

Determinations of geniposide using LC/MS/MS methods via forming ammonium and acetate adducts

In this paper, we report method development work to determine geniposide using LC/MS/MS via the formation of positive and negative ion adducts. Geniposide, which has been recognized to have choleretic effects, is the major iridoid glycoside component of Gardenia herbs. To enhance the sensitivity of LC/MS detection of geniposide, a small amount of volatile additives such as ammonium acetate and acetic acid are added into mobile phase solvents to form positive and negative adducts, which can then ionize via electrospray processes. The formation of positive adducts is due to the complexation between geniposide and ammonium ions ([M + NH₄]⁺). The formation of anionic adducts [M + CH₃COO]⁻ is believed to occur via hydrogen bonds bridging acetate ions and glucose groups on the geniposide molecule. Mobile phase solvents containing acetonitrile and aqueous solution (0.2 mM ammonium acetate or 0.1% acetic acid) at the ratio 15: 85 are employed to elute geniposide using C8 reverse phase liquid chromatography columns with electrospray tandem mass spectrometry determinations. Using geniposide standards, the methods are validated at the concentration ranges of 5 to 1000 ng/mL and 20 to 5000 ng/mL using ammonium and acetate adducts respectively. The correlation coefficients of the standard curves are 0.9999 using both ammonium and acetate adducts. The detection limits of using ammonium and acetate adducts are 1 and 5 ng/mL respectively. The measurement accuracy and precision of using ammonium adducts are within 12% and 3% respectively, whereas the accuracy and precision are within 6 and 11% respectively using acetate adducts. When the validated calibration curves of the ammonium adduct of geniposide are used to determine spiked control samples in rat blood dialysates, the determination errors of accuracy and precision are within 12% and 10% respectively.     

Mass fragmentographic determination of prostaglandin F2 alpha in human and rabbit urine

Analysis of prostaglandin F2 alpha (PGF2 alpha) in urine is a useful indicator of renal prostaglandin synthesis. A mass fragmentographic method for PGF2 alpha analysis in human urine was developed using [3,3,4,4-2H4]PGF2 alpha as an internal standard and carrier. PGF2 alpha was extracted from urine (20 ml) with chloroform, purified by preparative thin-layer chromatography and converted to the methyl ester trimethylsilyl ether before analysis by gas chromatography--mass spectrometry. The specificity of the urine analysis was demonstrated by retention time and the use of two pairs of fragments m/e 494/498 and 513/517 with the same results. The coefficient of variation for duplicate analysis averaged 12.6%, n = 17. Urine from recumbent women contained 4.9 +/- 2.6 (S.D.) ng/ml or 4.1 +/- 1.0 ng PGF2 alpha per mg creatinine (n = 10) with little diurnal variation. Male urine contained 5.0 +/- 2.7 (S.D.) ng/ml or 3.7 +/- 2.1 ng/mg creatinine (n = 10). Similar concentrations were found in boys and in girls. These observations indicate that urinary PGF2 alpha originates from the kidneys with little contribution from the male accessory sexual glands. This method can also be applied to analysis of PGF2 alpha in rabbit urine.     

Determination of prostaglandins and thromboxane in whole blood by high-performance liquid chromatography with fluorimetric detection

A highly sensitive and specific assay for the quantitation of prostaglandins (PGs) such as PGE1, PGE2, PGF1 alpha, PGF2 alpha, 6-keto-PGF1 alpha, and including thromboxane B2, is described. The method involves the addition of PGF1 alpha and PGE1 as the internal standards, extraction from whole blood and purification by silica gel column chromatography. Following conversion into the methoximes, purification by reversed-phase chromatography and esterification with panacyl bromide, samples are analysed by high-performance liquid chromatography with fluorimetric detection. The lower limit of detection of the eicosanoids 6-keto-PGF1 alpha, thromboxane B2 and PGF2 alpha in blood is ca. 50 pg/ml and that of PGE2 is 100 pg/ml. Assay linearity is demonstrated over a range from 60 pg to 60 ng of eicosanoid injected. The method allows simultaneous assessment of prostaglandins and thromboxane extracted from complex biological fluids at picogram levels.     

A validated LC–MS/MS method for quantitative determination of L-dopa in Fagioli di Sarconi beans (Phaseolus vulgaris L.)

An analytical method based on ultrasound assisted extraction (UAE) and liquid chromatography coupled to electrospray tandem mass spectrometry (LC–ESI/MS/MS) was validated and applied for determining L-dopa in four ecotypes of Fagioli di Sarconi beans (Phaseolus vulgaris L.), marked with the European label PGI (Protected Geographical Indication). The selectivity of the proposed method was ensured by the specific fragmentation of the analyte. Simple isocratic chromatographic conditions and mass spectrometric detection in multiple reaction monitoring (MRM) acquisition mode were used for sensitive quantification. The LC–ESI/MS/MS method was validated within a linear range of 0.001–5.000 μg/mL. Values of 0.4 and 1.1 ng/mL were obtained for the limits of detection and quantification, respectively. The repeatability, inter-day precision, and recovery values ranges were 0.6%–4.5%, 5.4%–9.9%, and 83%–93%, respectively. Fresh and dried beans, as well as pods, cultivated exclusively with organic methods avoiding any synthetic fertilizers and pesticides were analyzed showing an L-dopa content ranging from 0.020 ± 0.005 to 2.34 ± 0.05 μg/g dry weight.     

Simultaneous determination of prostanoids in plasma by gas chromatography-negative-ion chemical-ionization mass spectrometry

A method for simultaneous determination of prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha), 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), and thromboxane B2 (TxB2) in plasma was developed. After acidification and addition of 2H- and 3H-labelled internal standards, plasma prostanoids were extracted by reversed-phase cartridges and purified by normal-phase high-performance liquid chromatography. The pentafluorobenzyl, methoxime, trimethylsilyl derivatives were formed. Negative-ion chemical-ionization mass spectra with methane as reagent gas show one intense peak at m/z (M - pentafluorobenzyl). This ion was used for selective-ion monitoring. Prostanoid plasma concentrations (pg/ml) in five healthy volunteers were: PGE2 2.0-10.4, PGF2 alpha 2.2-9.8, 6-keto-PGF1 alpha 0.6-1.8, and TxB2 3.0-45.3. However, there is evidence that the TxB2 values may frequently be falsely high because of ex vivo production during the sampling procedure.     

Quantitation of melatonin and n‐acetylserotonin in human plasma by nanoflow LC‐MS/MS and electrospray LC‐MS/MS

Melatonin (MEL) and its chemical precursor N‐acetylserotonin (NAS) are believed to be potential biomarkers for sleep‐related disorders. Measurement of these compounds, however, has proven to be difficult due to their low circulating levels, especially that of NAS. Few methods offer the sensitivity, specificity and dynamic range needed to monitor MEL and its precursors and metabolites in small blood samples, such as those obtained from pediatric patients. In support of our ongoing study to determine the safety, tolerability and PK dosing strategies for MEL in treating insomnia in children with autism spectrum disorder, two highly sensitive LC‐MS/MS assays were developed for the quantitation of MEL and precursor NAS at pg/mL levels in small volumes of human plasma. A validated electrospray ionization (ESI) method was used to quantitate high levels of MEL in PK studies, and a validated nanospray (nESI) method was developed for quantitation of MEL and NAS at endogenous levels. In both assays, plasma samples were processed by centrifugal membrane dialysis after addition of stable isotopic internal standards, and the components were separated by either conventional LC using a Waters SymmetryShield RP18 column (2.1 × 100 mm, 3.5 µm) or on a polyimide‐coated, fused‐silica capillary self‐packed with 17 cm AquaC18 (3 µm, 125 Å). Quantitation was done using the SRM transitions m/z 233 → 174 and m/z 219 → 160 for MEL and NAS, respectively. The analytical response ratio versus concentration curves were linear for MEL (nanoflow LC: 11.7–1165 pg/mL, LC: 1165–116500 pg/mL) and for NAS (nanoflow LC: 11.0–1095 pg/mL). Copyright © 2012 John Wiley & Sons, Ltd.