A simple and fast LC–MS/MS method for the simultaneous determination of tenofovir alafenamide and tenofovir in human plasma

A simple and fast liquid chromatography–tandem mass spectrometry method was established and validated for the simultaneous determination of tenofovir alafenamide (TAF) and tenofovir (TNF) in human plasma. A simple protein precipitation procedure was employed to extract analytes from plasma. Chromatographic separation was performed on an Eclipse Plus C₁₈ column utilizing a fast gradient elution starting with 2% of 2 mM ammonium acetate–formic acid (100/0.1, v/v) followed by increasing the percentage of acetonitrile. Detection was performed on a tandem mass spectrometer equipped with an electrospray ionization source operated in the positive ionization mode, using the transitions m/z 477.2 → m/z 346.1 for TAF and m/z 288.1 → m/z 176.1 for TNF. TAF-d₅ and TNF-d₇ were used as the internal standard of TAF and TNF, respectively. The method was validated in the concentration ranges 1.25–500 ng/mlfor TAF and 0.300–15.0 ng/ml for TNF with acceptable accuracy and precision.     

Development and validation of a high-performance liquid chromatography-electrospray ionization-MS/MS method for the simultaneous quantitation of levodopa and carbidopa in human plasma

A sensitive and fast high‐performance liquid chromatography–electrospray ionization–MS/MS method for the simultaneous quantitation of levodopa and carbidopa in human plasma was developed and validated. A simple protein precipitation step with perchloric acid was used for the cleanup of plasma, and methyldopa was added as an internal standard. The analyses were carried out using an ACE C₁₈ column (50 × 4.6 mm i.d.; 5 µm particle size) and a mobile phase consisting of 0.2% formic acid and acetonitrile (90:10). The triple‐quadrupole mass spectrometer equipped with an electrospray source in positive mode was set up in the selective reaction monitoring mode to detect the ion transitions m/z 198.1 → m/z 107.0, m/z 227.2 → m/z 181.0, and m/z 212.1 → m/z 139.2 for levodopa, carbidopa, and methyldopa, respectively. The method was validated and proved to be linear, accurate, and precise over the range 50–5000 ng/mL for levodopa and 3–600 ng/mL for carbidopa. The proposed method was successfully applied in a pharmacokinetic study with a levodopa/carbidopa tablet formulation in healthy volunteers. Copyright © 2011 John Wiley & Sons, Ltd.     

Simultaneous determination of three flavonoids and one coumarin by LC–MS/MS: Application to a comparative pharmacokinetic study in normal and arthritic rats after oral administration of Daphne genkwa extract

A selective and sensitive liquid chromatography–tandem mass spectrometry method was developed and validated for investigating the pharmacokinetics of umbelliferone, apigenin, genkwanin and hydroxygenkwanin after oral administration of Daphne genkwa extract. Plasma samples were treated by protein precipitation with acetonitrile. Analytes were detected by triple‐quadrupole MS/MS with an ESI source in negative selection reaction monitoring mode. The transitions of m/z 161 → 133 for umbelliferone, m/z 269 → 117 for apigenin, m/z 283 → 268 for genkwanin and m/z 299 → 284 for hydroxygenkwanin were confirmed for quantification. Chromatographic separation was conducted using an Eclipse XDB‐C₁₈ column, and the applied isocratic elution program allowed for simultaneous determination of the four analytes for a total run time of 2.5 min. The linearity was validated over the plasma concentration ranges of 1.421–1421 ng/mL for umbelliferone, 0.845–845 ng/mL for apigenin, 1.025–1025 ng/mL for genkwanin and 0.845–845 ng/mL for hydroxygenkwanin. The extraction recovery rate was >82.7% for each analyte. No apparent matrix effect was observed during the bioanalysis. After full validation, the proposed method was successfully applied to compare the pharmacokinetics of these analytes between normal and arthritic rats.     

Rapid and robust confirmation and quantification of 11‐nor‐Δ9‐tetrahydrocannabinol‐9‐carboxylic acid (THC‐COOH) in urine by column switching LC‐MS‐MS analysis

A method for the rapid and robust confirmation of 11‐nor‐?9‐tetrahydrocannabinol‐9‐carboxylic acid (THCA) in urine involving basic hydrolysis with NaOH and direct injection of the hydrolysate in a column‐switching LC‐MS‐MS system was developed and validated. THCA‐d3 was used as internal standard. Detection was performed in negative‐ion mode by monitoring the transitions from the [M‐CO₂]‐ ion m/z 299.2→245.2 and and m/z 299.2→191.1 that were found to provide a better signal‐to‐noise ratio than the transition from the pseudomolecular ion at m/z 343. The high sensitivity of detection enabled the injection of a small volume (10 µl) of the NaOH hydrolysate which, together with the applied column switching system, proved to confer ruggedness to the method and to avoid the deterioration of the instrumental apparatus despite the large amount of inorganic ions in the hydrolysate. The LLOQ was established at 5 ng/ml, and the LLOD was calculated as 0.2 ng/ml (S/N =3). The method was submitted to thorough validation including evaluation of the calibration range (5–500 ng/ml), accuracy and precision, matrix effects, overall process efficiency, autosampler stability, carryover and cross‐talk, and 10‐times reduction of sample volume (0.1 ml). Proof of applicability was obtained by direct comparison with the reference GC‐MS method in use in the lab (the R² between the two methods was 0.9951). Copyright © 2012 John Wiley & Sons, Ltd.     

Simultaneous quantification of metformin and glipizide in human plasma by high‐performance liquid chromatography–tandem mass spectrometry and its application to a pharmacokinetic study

A selective, sensitive and rapid high‐performance liquid chromatography–tandem mass spectrometry (HPLC‐MS/MS) method was developed and validated to determine metformin and glipizide simultaneously in human plasma using phenacetin as internal standard (IS). After one‐step protein precipitation of 200 μL plasma with methanol, metformin, glipizide and IS were separated on a Kromasil Phenyl column (4.6 × 150 mm, 5 µm) at 40°C with an isocratic mobile phase consisting of methanol–10 mmol/L ammonium acetate (75:25, v/v) at a flow rate of 0.35 mL/min. Electrospray ionization source was applied and operated in the positive mode. Multiple reaction monitoring using the precursor → product ion combinations of m/z 130 → m/z 71, m/z 446 → m/z 321 and m/z 180 → m/z 110 were used to quantify metformin, glipizide and IS, respectively. The linear calibration curves were obtained over the concentration ranges 4.10–656 ng/mL for metformin and 2.55–408 ng/mL for glipizide. The relative standard deviation of intra‐day and inter‐day precision was below 10% and the relative error of accuracy was between ?7.0 and 4.6%. The presented HPLC‐MS/MS method was proved to be suitable for the pharmacokinetic study of metformin hydrochloride and glipizide tablets in healthy volunteers after oral administration. Copyright © 2012 John Wiley & Sons, Ltd.     

Development of a simple HPLC–MS/MS method to simultaneously determine teriflunomide and its metabolite in human plasma and urine: Application to clinical pharmacokinetic study of teriflunomide sodium and leflunomide

A simple high‐performance liquid chromatography coupled with tandem mass spectrometry method was developed and fully validated to simultaneously determine teriflunomide (TER) and its metabolite 4‐trifluoro‐methylaniline oxanilic acid (4‐TMOA) in human plasma and urine. Merely 50 μL plasma and 20 μL urine were employed in sample preparation using protein precipitation and direct dilution method, respectively. An Agilent Zorbax eclipse plus C₁₈ column was selected to achieve rapid separation for TER and 4‐TMOA within 3 min. Electrospray ionization under multiple reaction monitoring was used to monitor the ion transitions for TER (m/z 269.0 → 159.9), 4‐TMOA (m/z 231.9 → 160.0), internal standard teriflunomide‐d4 (m/z 273.0 → 164.0) and 2‐amino‐4‐trifluoromethyl benzoic acid (m/z 203.8 → 120.1), operating in the negative ion mode. This method proved to have better accuracy and precision over concentration range of 10–5000 ng/mL in plasma as well as 10–10,000 ng/mL in urine. After a full validation, this method was successfully applied in a pharmacokinetic study of teriflunomide sodium and leflunomide in Chinese healthy volunteers.     

Determination of rhynchophylline and hirsutine in rat plasma by UPLC‐MS/MS after oral administration of Uncaria rhynchophylla extract

An ultra‐performance liquid chromatography with tandem mass spectrometry (UPLC–MS/MS) method was developed and validated to concurrently determine rhynchophylline and hirsutine in rat plasma. The sample preparation of rat plasma was achieved by alkalization and liquid–liquid extraction. The mass transition of precursor ion → product ion pairs were monitored at m/z 385.2 → 160.0 for rhynchophylline, m/z 369.3 → 144.0 for hirsutine and m/z 414.0 → 220.0 for noscapine (internal standard). This method revealed linear relationships from 2.5 to 50 ng/mL (r² > 0.997) for rhynchophylline and from 2.5 to 50 ng/mL (r² > 0.998) for hirsutine. The limit of quantification values for rhynchophylline and hirsutine in rat plasma were both 2.5 ng/mL. Intra‐day and inter‐day precisions were within 10.6% and 12.5%, respectively, for rhynchophylline and hirsutine, and the accuracy (bias) was <10%. Liquid–liquid extraction of rat plasma samples resulted in insignificant matrix effect, and the extraction recoveries were >83.6% for rhynchophylline, 73.4% for hirsutine and 90.7% for the internal standard. This method was applied successfully to a pharmacokinetic study of rhynchophylline and hirsutine in rats after oral administration. Copyright © 2013 John Wiley & Sons, Ltd.     

Validated LC–MS/MS method for the simultaneous determination of enalapril maleate,nitrendipine, hydrochlorothiazide,and their major metabolites in human plasma

Hypertension is a major risk factor for atherosclerosis and ischemic heart disease. Most hypertensive patients need a combination of antihypertensive agents to achieve therapeutic goals. A rapid, sensitive, and selective liquid chromatography-tandem mass spectrometric method was developed and validated for simultaneous determination of enalapril maleate (ENA) and its major metabolite enalaprilat (ENAT), nitrendipine (NIT) and its major metabolite dehydronitrendipine (DNIT), and hydrochlorothiazide (HCT) in human plasma using felodipine as an internal standard (IS). The drugs were extracted from plasma using one-step protein precipitation. Chromatographic separation was performed on a Symmetry C₁₈ column, with water and acetonitrile (10:90, v/v) as mobile phase. The detection was carried out using multiple reaction monitoring mode and coupled with electrospray ionization source. Multiple reaction monitoring transitions were m/z 377.1 → 234.1 for ENA, m/z 349.2 → 206.1 for ENAT, m/z 361.2 → 315.1 for NIT, m/z 359 → 331 for DNIT, m/z 295.9 → 205.1 for HCT, and m/z 384.1 → 338 for felodipine (IS). The method was linear over concentration ranges of 1–200, 20–500, 5–200, 2–100, and 5–200 ng/mL for ENA, ENAT, NIT, DNIT, and HCT, respectively, with r² ≥ 0.99. Method validation was performed according to U.S. Food and Drug Administration guidelines. The validated method showed good sensitivity and selectivity and could be applied for therapeutic drug monitoring and bioequivalence studies.     

Simultaneous quantification of hyperin,reynoutrin and guaijaverin in mice plasma by LC‐MS/MS: application to a pharmacokinetic study

A specific and sensitive LC‐MS/MS assay was developed to simultaneously quantify three structurally similar flavonoid glycosides – hyperin, reynoutrin and guaijaverin – in mouse plasma. Biosamples were prepared by solid‐phase extraction. Isocratic chromatographic separation was performed on an AichromBond‐AQ C₁₈ column (250 × 2.1 mm, 5 μm) with methanol–acetonitrile–water–formic acid (20:25:55:0.1) as the mobile phase. Detection of hyperin, reynoutrin, guaijaverin and internal standard [luteolin‐7‐O‐β‐d ‐apiofuranosyl‐(1 → 6)‐β‐d ‐glucopyranoside] was achieved by ESI‐MS/MS in the negative ion mode using m/z 463 → m/z 300, m/z 433 → m/z 300, m/z 433 → m/z 300 and m/z 579 → m/z 285 transitions, respectively. Linear concentration ranges of calibration curves were 4.0–800.0 ng/mL for hyperin and reynoutrin and 8.0–1600.0 ng/mL for guaijaverin when 100 μL of plasma was analyzed. We used this validated method to study the pharmacokinetics of hyperin, reynoutrin and guaijaverin in mice following oral and intravenous administration. All three quercetin‐3‐O‐glycosides showed poor oral absorption in mice, and the absolute bioavailability of hyperin after oral administration of 100 mg/kg was 1.2%. Pretreatment with verapamil increased the peak concentration and area under the concentration–time curve of hyperin, which were significantly higher than the control values. The half‐life of hyperin with verapamil was significantly prolonged compared with that of the control. Copyright © 2016 John Wiley & Sons, Ltd.     

Development and validation of a sensitive and specific LC–MS/MS cocktail assay for CYP450 enzymes: Application to study the effect of catechin on rat hepatic CYP activity

A sensitive and specific liquid chromatography tandem mass spectrometric (LC–MS/MS) method that enables the simultaneous quantification of probe substrates and metabolites of cytochrome P450 (CYP) enzymes was developed and validated. These substrates (metabolites)—coumarin (7-hydroxycoumarin), tolbutamide (4-hydroxytolbutamide), S-mephenytoin (4-hydroxymephenytoin), dextromethorphan (dextrorphan), and testosterone (6β-hydroxytestosterone)—were utilized as markers for the activities of the major human CYP enzymes CYP2A6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, respectively. Analytes were separated on Kinetex C₁₈ column (2.1 × 50 mm, 5 μm) using a binary gradient mobile phase of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Metabolites were detected and quantified by MS using multiple reaction monitoring at m/z 163 → 107.2 for 7-hydroxycoumarin, m/z 235 → 150.1 for 4-hydroxymephenytoin, m/z 287 → 171 for 4-hydroxytolbutamide, m/z 258 → 157.1 for dextrorphan, m/z 305 → 269 for 6β-hydroxytestosterone, and m/z 237 → 194 for the internal standard. The assay exhibited good linearity over a range of 10–500 ng/mL with acceptable accuracy and precision criteria. As a proof of concept, the developed cocktail assay was successfully used to examine the potential impact of catechin on the activity of the major rat liver CYP enzymes.     

LC‐APCI mass spectrometric method development and validation for the determination of atovaquone in human plasma

A newly developed LC—APCI mass spectrometric method is described for human plasma determination of atovaquone using lapachol internal standard. A single‐step protein precipitation technique for plasma extraction of atovaquone achieving mean recovery of 94.17% (CV 8%) without compromising sensitivity (limit of quantitation 50.3 ng/mL) or linearity (50.3 ng/mL—23924.6 ng/mL) is delineated in this paper. Heated nebulizer in negative multiple reaction monitoring mode was employed with transitions m/z 365.2 → m/z 337.1 and m/z 240.9 → m/z 185.7 for atovaquone and lapachol respectively in this liquid chromatographic–tandem mass spectrometric method. Excellent chromatographic separation on a Synergi 4 μ Polar‐RP 80A (150 × 2.0 mm) column, using 100 μL of plasma extraction volume along with 10 μL of injection load, completing analysis run‐time within 2.5 min, highlights this simple yet unique bioanalytical method. The developed method can be successfully applied to pharmacokinetic studies on atovaquone suspension administered in healthy volunteers or HIV‐infected patients. Moreover full method validation results not published before are presented and discussed in detail for the first time in this article. Copyright © 2009 John Wiley & Sons, Ltd.     

Validation and application of a novel UHPLC–MS/MS method for the measurement of furanodienone in rat plasma

A sensitive ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was established to analyze furanodienone in rat plasma. In the process of chromatographic separation, selected reaction monitoring transitions for furanodienone and patchouli alcohol (internal standard, IS) were m/z 231.1 → 83.2 and m/z 205.1 → 95.1, respectively. Great linearity of furanodienone in plasma samples was found in the corresponding concentration range (r > 0.995). Intra- and inter-day precisions (RSD, %) were <11.3% in plasma, and the accuracy (RE, %) was within ±10.7%. This method was used to the furanodienone study on rat pharmacokinetics after a single oral dose of 10 mg/kg of furanodiene. The results indicated that the maximum observed plasma concentration was 52.4 ± 19.1 ng/ml at 1.2 ± 0.7 h with an elimination half-life of 2.2 ± 0.7 h. The obtained data indicated that furanodienone could be moderately distributed and eliminated.     

Determination of four antiepileptic drugs in plasma using ultra‐performance liquid chromatography with mass detection technique

Status epilepticus (SE) is considered the second most frequent neurological emergency. Its therapeutic management is performed using sequential antiepileptic drug regimens. Diazepam (DIA), midazolam (MID), phenytoin (PHT) and phenobarbital (PB) are four drugs of different classes used sequentially in the management of SE. A sensitive, selective, accurate and precise method was developed and validated for simultaneous determination of the four antiepileptic drugs in human plasma. Their separation and quantification were achieved using ultra‐performance liquid chromatography (UPLC) with mass detection using carbamazepine as internal standard (IS). For the first three drugs and the IS, UPLC–MS/MS with electrospray ionization working in multiple reaction monitoring mode was used at the following transitions: m/z 285 → 193 for DIA; m/z 326 → 291 for MID; m/z 253 → 182 for PHT; and m/z 237 → 194, 237 → 192 for IS. For the fourth drug (PB), a molecular ion peak of PB [M + H] ⁺ at m/z 233 was used for its quantitation. The method was linear over concentration ranges 5–500 ng/mL for DIA and MID and 0.25–20 μg/mL for PHT and PB. Bioanalytical validation of the developed method was carried out according to European Medicines Agency guidelines. The developed method can be applied for routine drug analysis, therapeutic drug monitoring and bioequivalence studies.     

Quantitation of glucosamine sulfate in plasma by HPLC-MS/MS after administration of powder for oral solution formulation

A rapid method for the quantification of glucosamine in human plasma using high‐performance liquid chromatography coupled to tandem mass spectrometry was developed and validated. The sample preparation includes a simple deproteinization step, using d ‐[1‐¹³C] glucosamine hydrochloride as an internal standard. Chromatographic separation was performed on an ACE Ciano column using isocratic elution with acetonitrile and aqueous 2 mm ammonium acetate containing 0.025% formic acid (80:20). Selected reaction monitoring was performed using the transitions m/z 180.1 → m/z 72.1 and m/z 181.0 → m/z 74.6 to quantify glucosamine and internal standard, respectively. The method was validated and proved to be linear, accurate and precise over the range 50–5000 ng/mL of glucosamine. Recovery rates higher than 90% were obtained for both glucosamine and internal standard. No matrix effect was detected in the samples. The validated method was successfully applied to a pharmacokinetic study after oral administration of a powder for oral solution formulation containing glucosamine sulfate. Copyright © 2011 John Wiley & Sons, Ltd.     

Simultaneous determination of evodiamine and its four metabolites in rat plasma by LC–MS/MS and its application to a pharmacokinetic study

A simple and sensitive liquid chromatography tandem mass spectrometry method was validated for simultaneous quantification of evodiamine and its metabolites 10‐hydroxyevodiamine (M1), 18‐hydroxyevodiamine (M2), 10‐hydroxyevodiamine‐glucuronide (M3) and 18‐hydroxy‐ evodiamine‐glucuronide (M4) in rat plasma for the first time. The analytes were extracted with acetonitrile and separated on a C₁₈ column within 3 min. The detection was achieved in positive selected reaction monitoring mode with precursor‐to‐product transitions at m/z 304.1 → 161.1 for evodiamine, m/z 320.1 → 134.1 for M1, m/z 320.1 → 150.1 for M2, m/z 496.2 → 134.1 for M3, m/z 496.2 → 171.1 for M4 and m/z 349.2 → 305.1 for camptothecin (internal standard). The linearity was evident over the tested concentration ranges with correlation coefficients >0.9991. The lower limits of quantification for evodiamine, M1, M2, M3 and M4 were 0.1, 0.1, 0.1, 0.25 and 0.25 ng mL⁻¹, respectively. Extraction recoveries and matrix effects of the analytes were within the ranges of 84.51–97.21 and 90.13–103.30%, respectively. The accuracy (relative error) ranged from −8.14 to 7.23% while the intra‐ and inter‐day precisions (relative standard deviation) were < 9.31%. The validated assay was successfully applied for the pharmacokinetic study of evodiamine, M1, M2, M3 and M4 in rat. The current study will be helpful in understanding the in vivo disposition of evodiamine.     

Development,validation and application of a UHPLC–MS/MS method for quantification of the adiponectin-derived active peptide ADP355 in rat plasma

A UHPLC–MS/MS method for the quantification of ADP355, an adiponectin-derived active peptide, was developed and validated. The extraction method employed simple protein precipitation using methanol and chromatographic separation was achieved on anAccucore™ RP-MS C₁₈ column (100 × 2.1 mm, 2.6 μm, 80 Å), using 0.1% formic acid in both water and acetonitrile with gradient elution at the flow rate of 400 μl/min within 4.0 min. Detections were performed under positive ion mode with multiple reaction monitoring ion transitions m/z 1109.2 → 309.8 and 871.4 → 310.1 for ADP355 and Jt003 respectively at unit resolution. The linearity range of the calibration curve was 2–1,000 ng/ml with a lower limit detection of 0.5 ng/ml. The selectivity, linearity, precision, accuracy, recovery, matrix effect and stability were validated, and all items met the requirement of US Food and Drug Administration guidance. This method was successfully applied to an intravenous pharmacokinetic study of ADP355 in rats and the in-vitro stability in rat serum, plasma and whole blood was also assessed.     

A validated LC–MS/MS method for the simultaneous determination of thalidomide and its two metabolites in human plasma: Application to a pharmacokinetic assay

An accurate and sensitive LC–MS/MS method for determining thalidomide, 5‐hydroxy thalidomide and 5′‐hydroxy thalidomide in human plasma was developed and validated using umbelliferone as an internal standard. The analytes were extracted from plasma (100 μL) by liquid–liquid extraction with ethyl acetate and then separated on a BETASIL C₁₈ column (4.6 × 150 mm, 5 μm) with mobile phase composed of methanol–water containing 0.1% formic acid (70:30, v/v) in isocratic mode at a flow rate of 0.5 mL/min. The detection was performed using an API triple quadrupole mass spectrometer in atmospheric pressure chemical ionization mode. The precursor‐to‐product ion transitions m/z 259.1 → 186.1 for thalidomide, m/z 273.2 → 161.3 for 5‐hydroxy thalidomide, m/z 273.2 → 146.1 for 5′‐hydroxy thalidomide and m/z 163.1 → 107.1 for umbelliferone (internal standard, IS) were used for quantification. The calibration curves were obtained in the concentrations of 10.0–2000.0 ng/mL for thalidomide, 0.2–50.0 ng/mL for 5‐hydroxy thalidomide and 1.0–200.0 ng/mL for 5′‐hydroxy thalidomide. The method was validated with respect to linear, within‐ and between‐batch precision and accuracy, extraction recovery, matrix effect and stability. Then it was successfully applied to estimate the concentration of thalidomide, 5‐hydroxy thalidomide and 5′‐hydroxy thalidomide in plasma samples collected from Crohn's disease patients after a single oral administration of thalidomide 100 mg.     

Pharmacokinetics and tissue distribution study of bisabolangelone from Angelicae Pubescentis Radix in rat using LC–MS/MS

A sensitive and accurate LC–MS/MS method was established for quantifying bisabolangelone in rat plasma and tissues. Bisabolangelone was isolated and purified from Angelicae Pubescentis Radix. The pharmacokinetic and tissue distribution of bisabolangelone after administration to rat was performed by LC–MS/MS. Separation was carried out on a C₈ (4.6 × 100 mm, 1.8 μm) column. The MS/MS transitions of bisabolangelone and tussilagone (internal standard) were set at m/z 249.1 → 109.1 and m/z 391.4 → 217.4, respectively. The lower limit of quantification in plasma and other tissues ranged from 1 to 4 ng/mL. The biosamples were prepared using protein precipitation method with acetonitrile. The recovery was >92%. The results showed that values of maximum concentrations and area under the curve depended linearly on the studied doses (2.5, 5 and 7.5 mg/kg body weight). The other ingredients in Angelicae Pubescentis Radix extract possibly reduce the absorption of bisabolangelone in rat. Tissue distribution revealed that bisabolangelone was widely distributed in vivo. The highest and lowest concentrations of bisabolangelone were found in the stomach and in the brain, respectively. It was concluded that the newly established HPLC–MS/MS method was suitable to describe the pharmacokinetic characteristics of bisabolangelone in rat after administration.     

Simultaneous determination of nicotinamide and N1-methylnicotinamide in human serum by LC–MS/MS to associate their serum concentrations with obesity

A rapid and sensitive LC–MS/MS method was developed and validated for the simultaneous determination of nicotinamide and its metabolite N¹-methylnicotinamide in human serum. Serum samples were prepared by protein precipitation with acetonitrile. The chromatographic separation was performed on a Waters Spherisorb S5 CN microbore column (2.0 × 100 mm, 5 μm) with gradient elution within 7 min. Acetonitrile and 5 mm ammonium formate aqueous solution (containing 0.1% formic acid) were used as mobile phases. Nicotinamide, N¹-methylnicotinamide and N′-methylnicotinamide (internal standard) were detected with a triple-quadrupole tandem mass spectrometer in the positive ion mode. Multiple reaction monitoring was used to monitor precursor to product ion transitions of m/z 123.1 → 80.1 for nicotinamide, m/z 137.1 → 94.1 for N¹-methylnicotinamide and m/z 137.1 → 80.1 for the internal standard. The linear ranges of nicotinamide and N¹-methylnicotinamide were 5.000–160.0 and 2.500–80.00 ng/ml, respectively. The intra- and inter-day precisions (RSD) of both analytes were within 6.90%. The recoveries were >88%. The analytes were proven to be stable during all sample storage, preparation and analytic procedures. The method was successfully applied to determine the concentrations of nicotinamide and N¹-methylnicotinamide in human serum to investigate the association between their concentrations and obesity in 1160 Chinese subjects.     

An LC‐MS/MS method for determination of 3,6′‐disinapoylsucrose in rat plasma and its application to a pharmacokinetic study

3,6′‐Disinapoylsucrose (DSS), a major active component of traditional Chinese medicine Yuan‐Zhi (the roots of Polygala tenuifolia), has significant effects for neuroprotection and improving learning memory. In order to explore the pharmacokinetic properties of DSS so as to further understand its in vivo activities, a sensitive LC‐MS/MS method was developed for determination of DSS in rat plasma and applied to a pharmacokinetic study in the present study. After treatment by protein precipitation, the plasma sample was separated on a C₁₈ HPLC column and analyzed by a mass spectrometry under positive electrospray ionization. Multiple‐reaction monitoring was employed to measure the ion transition at m/z 777.4 → 409.2 for DSS and m/z 557.2 → 309.1 for forsythin as internal standard. The method was linear over the studied concentration range of 0.5–1000.0 ng/mL. The precision and accuracy ranged from 1.4 to 18.4%, and from ?3.7 to ?9.5%, respectively, for within‐day and between‐day assay. Extraction recovery was higher than 86.6%. The limits of detection and quantification were 0.3 and 0.5 ng/mL, respectively. The present method was successfully applied to a pharmacokinetic study. DSS was found to have poor oral absorption with only about 0.5% bioavailability. Copyright © 2009 John Wiley & Sons, Ltd.