MS/MS in structural analysis of an oviposition-deterring pheromone

A recently characterized oviposition-deterring pheromone (ODP, structure 1) of the European cherry fruit fly was used as a test case for probing the potential of tandem mass spectrometry (MS/MS) in structure elucidation as a stand-alone technique. The glycolipid-taurinate 1 was subjected to MS/MS analyses under a variety of conditions with and without preceding chemical degradation. Acidic methanolysis of 1 and subsequent in-batch derivatization (trideuterioacetylation) yielded methyl 2,3,4,6-tetrakis-O-trideuterioacetyl-glucopyranoside (2), methyl 8,15-bis-trideuterioacetoxy-palmitate (3), and taurine (4) as suitable target compounds for direct mixture analysis.Low energy collision induced dissociation (CID) on selected precursor ions (MS/MS on [M + H – CH₃OH]⁺ and [M + H]⁺ produced by fast atom bombardment (FAB)) allowed direct identification of 2 and 4, respectively, by comparison with appropriate reference ions. In the case of 3, low energy CID (desorption chemical ionization (DCI) instead of FAB, MS/MS on [M + H]⁺) permitted deduction of gross molecular structure, but failed to provide positional detail. In sharp contrast,high energy CID of trideuterioacetylated intact 1 (FAB-MS/MS on [M – H]^– ions of la) clearly revealed a linear 8,15-hydroxylated palmitic acid backbone. Less certain was assignment of 15-O-glucosylation by this approach.     

LC‐MS/MS determination and pharmacokinetic study of lacidipine in human plasma

A robust, specific and fully validated LC‐MS/MS method as per general practices of industry has been developed for estimation of lacidipine (LAC) with 100 μL of human plasma using lacidipine‐¹³C8 as an internal standard (IS). The API‐4000 LC‐MS/MS was operated under the multiple reaction‐monitoring mode. A simple liquid–liquid extraction process was used to extract LAC and IS from human plasma. The total run time was 3.0 min and the elution of LAC and IS occurred at 1.96 and 1.97 min; this was achieved with a mobile phase consisting of 5 mm ammonium acetate buffer–acetontrile (15:85 v/v) at a flow rate of 0.60 mL/min on a Zorbax SB C₁₈ (50 × 4.6 mm, 5 µm) column. A linear response function was established for the range of concentrations 50–15,000 pg/mL (r > 0.998) for LAC. The current developed method has negligible matrix effect and is free from unwanted adducts and clusters which are formed owing to system such as solvent or mobile phase. The developed assay method was applied to an oral pharmacokinetic study in humans and successfully characterized the pharmacokinetic data up to 72 h. Copyright © 2013 John Wiley & Sons, Ltd.     

A mass spectrometric method to determine activities of enzymes involved in polyamine catabolism

An analytical method for the determination of three polyamines (putrescine, spermidine, and spermine) and five acetylpolyamines [N¹-acetylspermidine (N¹AcSpd), N⁸-acetylspermidine (N⁸AcSpd), N¹-acetylspermine, N¹,N⁸-diacetylspermidine, and N¹,N¹²-diacetylspermine] involved in the polyamine catabolic pathway has been developed using a hybrid tandem mass spectrometer. Heptafluorobutyryl (HFB) derivatives of these compounds and respective internal standards labeled with stable isotopes were analyzed simultaneously by TOF MS, based on peak areas appearing at appropriate m/z values. The isomers, N¹AcSpd and N⁸AcSpd were determined from their fragment ions, the acetylamidopropyl and acetylamidobutyl groups, respectively, using MS/MS with ¹³C₂-N¹AcSpd and ¹³C₂-N⁸AcSpd which have the ¹³C₂-acetyl group as an internal standard. The TOF MS method was successfully applied to measure the activity of enzymes involved in polyamine catabolic pathways, namely N¹-acetylpolyamine oxidase (APAO), spermine oxidase (SMO), and spermidine/spermine N¹-acetyltransferase (SSAT). The following natural substrates and products labeled with stable isotopes considering the application to biological samples were identified; for APAO, [4,9,12-¹⁵N₃]-N¹-acetylspermine and [1,4,8-¹⁵N₃]spermidine (¹⁵N₃-Spd), respectively; for SMO, [1,4,8,12-¹⁵N₄]spermine and ¹⁵N₃-Spd, respectively; and for SSAT, ¹⁵N₃-Spd and [1,4,8-¹⁵N₃]-N¹-acetylspermidine, respectively.     

ESI‐MS/MS analysis of protonated N‐methyl amino acids and their immonium ions

Methylation is one of the important posttranslational modifications of biological systems. At the metabolite level, the methylation process is expected to convert bioactive compounds such as amino acids, fatty acids, lipids, sugars, and other organic acids into their methylated forms. A few of the methylated amino acids are identified and have been proved as potential biomarkers for several metabolic disorders by using mass spectrometry–based metabolomics workstation. As it is possible to encounter all the N‐methyl forms of the proteinogenic amino acids in plant/biological systems, it is essential to have analytical data of all N‐methyl amino acids for their detection and identification. In earlier studies, we have reported the ESI‐MS/MS data of all methylated proteinogenic amino acids, except that of mono‐N‐methyl amino acids. In this study, the N‐methyl amino acids of all the amino acids ( 1 ‐ 21 ; including one isomeric pair) were synthesized and characterized by ESI‐MS/MS, LC/MS/MS, and HRMS. These data could be useful for detection and identification of N‐methyl amino acids in biological systems for future metabolomics studies. The MS/MS spectra of [M + H]⁺ ions of most N‐methyl amino acids showed respective immonium ions by the loss of (H₂O, CO). The other most common product ions detected were [MH‐(NH₂CH₃]⁺, [MH‐(RH)]⁺ (where R = side chain group) ions, and the selective structure indicative product ions due to side chain and N‐methyl group. The isomeric/isobaric N‐methyl amino acids could easily be differentiated by their distinct MS/MS spectra. Further, the MS/MS of immonium ions inferred side chain structure and methyl group on α‐nitrogen of the N‐methyl amino acids.     

LC/ESI‐MS/MS characterisation of lipopeptide biosurfactants produced by the Bacillus licheniformis V9T14 strain

Lipopeptide biosurfactants produced by the Bacillus licheniformis V9T14 strain showed an interesting anti‐adhesion activity against biofilm formation of human pathogenic bacterial strains. The chemical characterisation of the crude extract of V9T14 strain was first developed through electrospray ionisation mass spectrometry (ESI‐MS) and ESI‐MS/MS direct infusions: two sets of molecular ion species belonging to the fengycin and surfactin families were revealed and their structures defined, interpreting their product ion spectra. The LC/ESI‐MS analysis of the crude extract allowed to separate in different chromatogram ranges the homologues and the isoforms of the two lipopeptide families. The extract was then fractionated by silica gel chromatography in two main fractions, I and II. The purified biosurfactants were analysed through a new, rapid and suitable LC/ESI‐MS/MS method, which allowed characterising the composition and the structures of the produced lipopeptides. LC/ESI‐MS/MS analysis of fraction I showed the presence of C₁₃, C₁₄ and C₁₅ surfactin homologues, whose structures were confirmed by the product ion spectra of the sodiated molecules [M + Na]⁺ at m/z 1030, 1044 and 1058. LC/ESI‐MS/MS analysis of fraction II confirmed the presence of two main fengycin isoforms, with the protonated molecules [M + H]⁺ at m/z 1478 and 1506 corresponding to C₁₇ fengycin A and C₁₇ fengycin B, respectively. Other homologues (C₁₄ to C₁₆) were revealed and confirmed as belonging to fengycin A or B according to the retention times and the product ions generated, although with the same nominal mass. Finally, a relative percentage content of each homologue for both lipopeptides families in the whole extract was proposed. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of proline in human serum by a robust LC‐MS/MS method: application to identification of human metabolites as candidate biomarkers for esophageal cancer early detection and risk stratification

Altered serum proline levels are related to cancer metabolism. This study developed and validated a LC‐MS/MS method to analyze proline in human serum. Surrogate blank serum, coupled with stable isotope l ‐proline‐¹³C₅,¹⁵ N as internal standard, was used for generating standard curves ranging from 2.5 to 100 μg/mL. Proline was extracted from serum samples using methanol. A Phenomenex Lux 5u Cellulose‐1 column (250 × 4.6 mm) was used for chromatographic separation with 40% methanol in 0.05% formic acid aqueous solution as a mobile phase. Mass detection was performed under positive ionization electrospray. Intra‐ and inter‐day accuracy and precision were <10%. The extraction recovery and matrix factor were 99.17 and 1.47%, respectively. Our study showed that the chiral column had high specificity and selectivity for separating proline from serum components. The assay was successfully applied for the quantification of human serum proline levels from 30 esophageal cancer patients and 30 healthy volunteers. Statistical analyses showed significantly lower levels of serum proline in the patients as compared with the healthy volunteers (p‐value = 0.011). We report here a simple, specific and reproducible LC‐MS/MS method for the quantification of proline in human serum as a potential screening biomarker for esophageal cancer. Copyright © 2014 John Wiley & Sons, Ltd.     

Rapid Detection of the Residual Kryptofix 2.2.2 Levels in [18F]-Labeled Radiopharmaceuticals by Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry

A fast and sensitive ultra-performance liquid chromatography-tandem mass spectrometric (UPLC/MS/MS) method was developed and validated for determination of the residual levels of Kryptofix 2.2.2 (K222) in [¹⁸F]-labeled radiopharmaceuticals. The analytical time was only 3 min, and the injection volume was 5 μL. An electrospray ionization source was used in the positive mode (ESI⁺) for UPLC/MS/MS. The analytical measurements were performed in the multiple reaction monitoring (MRM) mode. The calibration curve at the spiked concentrations of 2–500 ng/mL for K222 showed good linearity. The intra- and inter-day precisions were not more than 5%. The accuracy satisfied the requirement of quality control analysis, the recoveries were found to be 80–120%. This method was successfully applied to detect the residue of K222 in [¹⁸F]-fluorodeoxyglucose [(¹⁸F)FDG], [¹⁸F]-fluoromisonizole[(¹⁸F)FMISO], 3′-deoxy-3′-[¹⁸F]-fluorothymidine [(¹⁸F)FLT], and two new [¹⁸F]-labeled radiopharmaceuticals 4-[-(2-[¹⁸F]fluoroethoxy) methyl]-1-[2-(2-methyl-5-nitro-1H- imidazol-1-yl) ethyl]-1H-1,2,3-triazole (named as ¹⁸F-BNU-1) and 4-[-(2-[¹⁸F] fluoroethoxy) methyl]-1-[2-(2-nitro-1H-imidazol-1-yl) ethyl]-1H-1,2,3-triazole (named as ¹⁸F-BNU-2) produced in our lab.     

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.     

The Synthesis of 13C9-15N-Labeled 3,5-Diiodothyronine and Thyroxine

Thyroid hormones undergo extensive metabolism to regulate hormone activity. A labeled thyroid hormone would be useful to track hormone metabolism through various pathways. While radiolabeled thyroid hormones have been synthesized and used for in vivo studies, a stable isotope labeled form of thyroid hormone is required for studying thyroid hormone metabolism by LC-MS/MS, an analytical technique that has certain advantages without the complications of radioactivity. Here we report the synthesis of ¹³C₉-¹⁵N-T₂ and ¹³C₉-¹⁵N-T₄, two labeled thyroid hormone derivatives suitable for in vivo LC-MS/MS studies.

Supplemental materials are available for this article. Go to the publisher's online edition of Synthetic Communications® to view the free supplemental file.     

Investigation of Reaction Mechanism of Amino Acids and Phosphorus Trichloride by 31P NMR and ESI‐MS/MS

The reaction of amino acids and phosphorus trichloride in THF was studied by ³¹P NMR tracing and ESI‐MS/MS. A series of hydridophoranes and cyclic dipeptides were obtained. The reaction presented interesting diversity and the reaction mechanism was proposed. The mechanism suggests that phosphorus plays an important role in the synthesis of amino acid hydridophorane and cyclic dipeptides. The results also show that ³¹P NMR and ESI‐MS/MS are useful tools for the investigation of reaction mechanism.     

Selectable one-dimensional or two-dimensional gas chromatography–mass spectrometry with simultaneous olfactometry or element-specific detection

A novel selectable one-dimensional (¹D) or two-dimensional (²D) gas chromatography–mass spectrometry (selectable ¹D/²D GC–MS) system with selective detection was developed by using capillary flow technology and low thermal mass GC (LTM-GC). The main advantages of this system are the simple and fast selection of ¹D GC–MS or ²D GC–MS operation without any instrumental set-up change (e.g.²D GC–MS can be run just after ¹D GC–MS run), and simultaneous mass spectrometric and olfactometry or element-specific detection for both ¹D and ²D separation to assure selection of a heart-cut region and correct identification of compounds of interest. The feasibility and benefit of the proposed system with selective detection, e.g. olfactometry, nitrogen phosphorus detection (NPD), and pulsed flame photometric detection (PFPD), was demonstrated with an identification of trace amounts of aroma components in beverages (beer and coffee). Using stir bar sorptive extraction (SBSE) and selectable ¹D/²D GC–Olfactometry/MS on a beer sample, β-damascenone could be determined at 1.9 ng mL⁻¹ (RSD 3.1%, n = 6) as a potent aroma compound. In a coffee sample, two odor active compounds were clearly resolved from a 4.2 s heart-cut and were assigned probable identifications as 4,5-dimethyl thiazole and dimethyl trisulfide based on a NIST library search, dual linear retention indices (dual LRI) and elemental information obtained by SBSE in combination with selectable ¹D/²D GC–NPD/PFPD/MS.     

Simultaneous determination of 103 pesticide residues in tea samples by LC‐MS/MS

A simple and sensitive method was developed and validated for the simultaneous determination of 103 pesticide residues in tea by LC‐MS/MS. For the analysis of the pesticide with polarity, thermal lability or low volatility, this LC‐MS/MS method has an advantage over GC. In this work, residual pesticides were extracted from the tea sample with ACN and then purified using Carb‐NH₂ SPE cartridges. Using the multiple reaction monitoring mode, the pesticides were quantified and identified by the most abundant and characteristic fragment ions. The recoveries obtained for each pesticide ranged between 65 and 114% at three spiked concentration levels. The intra‐day precisions were lower than 19.6%. Good linear relationships were observed with the correlation coefficients r² >0.996 for all analytes. The established method was successfully applied to the determination of pesticide residues in real tea samples.     

Screening of exon methylation biomarkers for colorectal cancer via LC‐MS/MS strategy

The identification of biomarkers would be of benefit for the diagnosis and treatment of colorectal cancer. DNA methylation in specific genomic regions, which had shown strongly association with disease genotypes, was an effective indicator to reveal the occurrence and development of cancers. To screen out methylation biomarkers for colorectal cancer (CRC), genomic DNA was isolated from colorectal cancerous and corresponding cancer‐adjacent tissues collected from 30 CRC patients and then bisulfite‐converted. The exon regions of 5 targeted genes (CNRIP1 , HIC1 , RUNX3 , p15 , and SFRP2 ) were amplified by using nested polymerase chain reaction with specific primers, and the amplicon was purified and hydrolyzed. The methylation levels of these specific regions were detected by liquid chromatography tandem mass spectrometry (LC‐MS/MS). The results showed that 5 targeted exon regions were successfully amplified and confirmed by sequencing. The methodological validations indicated that LC‐MS/MS was highly sensitive and accurate. The methylation levels of CNRIP1 and RUNX3 were remarkably high in CRC tissues with statistical difference when compared with corresponding cancer‐adjacent individuals, while that of HIC1 , p15 , and SFRP2 had no difference between 2 subjects. These findings supported CNRIP1 and RUNX3 as potential DNA methylation biomarkers for CRC diagnosis and treatment, and our LC‐MS/MS approach exhibited great advantages in the identification of regional DNA methylation biomarkers.     

Selected ion storage versus tandem MS/MS for organochlorine pesticides determination in drinking waters with SPME and GC-MS

The use of two modes for mass spectrometry (MS) detection with an ion trap instrument, selected ion storage (SIS) and tandem mass spectrometry (MS/MS), are compared for the solid-phase microextraction (SPME)–gas chromatography (GC) coupled to mass spectrometry (GC-MS) determination of 16 priority organochlorine pesticides (OCPs) in drinking water samples at the ultratrace levels (ng?L^?1) required by official guidelines in the European legislation. Experimental parameters investigated for the SPME sample preparation were: the type of coating (100?µm polydimethylsiloxane, PDMS, and 65?µm poly(dimethylsiloxane)–divinylbenzene, PDMS/DVB), SPME modality, extraction and desorption times and desorption temperature and the methanol percentage in the SPME working solution. Under the calculated optimal conditions two methodologies were developed, one for SIS and the other for MS/MS modes. The detection limits, precision and accuracy were evaluated for both alternatives and were appropriate to the official guidelines requirements. The SPME–GC-MS(SIS) methodology offered LODs from 0.2–6.6?ng?L^?1, precision below 13% and recoveries between 83 and 110%. The SPME–GC–MS/MS methodology provided limits of detection (LODs) ranging from 0.3 to 7.6 ng?L^?1, % RSD were ≤14% and recoveries of 79–108% were achieved. After the results observed within an Interlaboratory Exercise, the latest MS methodology was selected for the pursued analysis in real drinking water samples. Also, the good results in this round-robin exercise validate the proposed SPME–GC–MS/MS methodology.     

A rapid and sensitive determination of hypoxic radiosensitizer agent nimorazole in rat plasma by LC‐MS/MS and its application to a pharmacokinetic study

A highly sensitive, accurate and robust LC‐MS/MS method was developed and validated for determination of nimorazole (NMZ) in rat plasma using metronidazole (MNZ) as internal standard (IS). The analyte and IS were extracted from plasma by precipitating protein with acetonitrile and were chromatographed using an Agilent Poroshell 120, EC‐C₁₈ column. The mobile phase was composed of a mixture of acetonitrile and 0.1 % formic acid (85:15 v/v). The total run time was 1.5 min and injection volume was 5 μL. Multiple reaction monitoring mode using the transitions of m/z 227.1 → m/z 114.0 for MNZ and m/z 172.10 → m/z 128.1 for IS were monitored on a triple quadrupole mass spectrometer, operating in positive ion mode. The calibration curve was linear in the range of 0.25–200 ng/mL (r² > 0.9996) and the lower limit of quantification was 0.25 ng/mL in the rat plasma samples. Recoveries of NMZ ranged between 88.05 and 95.25%. The precision (intra‐day and inter‐day) and accuracy of the quality control samples were 1.25–8.20% and ?2.50–3.10, respectively. The analyte and IS were found to be stable during all sample storage and analysis procedures. The LC‐MS/MS method described here was validated and successfully applied to pharmacokinetic study in rats. Copyright © 2015 John Wiley & Sons, Ltd.     

A quantitative determination of fluorochloridone in rat plasma by UPLC‐MS/MS method: application to a pharmacokinetic study

A precise, high‐throughput and sensitive ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method has been developed for the determination of fluorochloridone (FLC) in rat plasma. The extraction of analytes from plasma samples was carried out by protein precipitation procedure using acetonitrile prior to UPLC‐MS/MS analysis. Verapamil was proved as a proper internal standard (IS) among many candidates. The chromatographic separation based on UPLC was well optimized. Multiple reaction monitoring in positive electrospray ionization was used with the optimized MS transitions at: m/z 312.0 → 292.0 for FLC and m/z 456.4 → 165.2 for IS. This method was well validated with good linear response (r² > 0.998) observed over the investigated range of 3–3000 ng/mL and with satisfactory stability. This method was also characterized with adequate intra‐ and inter‐day precision and accuracy (within 12%) in the quality control samples, and with high selectivity and less matrix effect observed. Total running time was only 1.5 min. This method has been successfully applied to a pilot FLC pharmacokinetic study after oral administration. Copyright © 2016 John Wiley & Sons, Ltd.     

A validated LC–MS/MS method for simultaneous quantification of simvastatin and simvastatin acid in beagle plasma: Application to an absolute bioavailability study

A highly sensitive LC–MS/MS method for simultaneous detection of both simvastatin (SV) and simvastatin acid (SVA) in beagle plasma was developed and successfully applied to an absolute bioavailability study. Lovastatin (LV) was used as internal standard (IS). The analysis was performed using electrospray ionization and selective reaction monitoring in positive mode at m/z 441.0 → 325.0 for SV, 459.0 → 343.0 for SVA and 427.0 → 325.0 for the IS, respectively. The assay procedure involved a simple liquid–liquid extraction of SV, SVA and LV from beagle plasma into methyl tert-butyl ether. Separation of SV, SVA and the IS was achieved on a Shim-pack VP-ODS column (150 × 2.0 mm, 5 μm) with a binary gradient solvent system of 0.1% formic acid in water and methanol (15:85, v/v) as the mobile phase. The method was validated over the range of 0.25–500 ng/ml for SV (r² ≥ 0.9923) and 0.24–481.23 ng/ml for SVA (r² ≥ 0.9987). The results of method validation for accuracy, precision, extraction recovery, matrix effect and stability were within the acceptance criteria. The values of absolute bioavailability of SV and SVA in beagles were 2.97 and 25.40%, respectively. It is the first study developed for the measurement of absolute bioavailability of SV and SVA acid in beagles.     

Simultaneous analysis of 35 specific antihypertensive adulterants in dietary supplements using LC/MS/MS

A sensitive and specific LC/MS/MS method was developed for the simultaneous analysis of 35 compounds used for treating hypertension as adulterants in dietary supplements. The method was validated for specificity, linearity, accuracy, precision, limit of detection, limit of quantitation, stability and recovery. The limit of detection and limit of quantitation ranged from 0.20 to 20.0 and 0.50 to 60.0 ng/g, respectively. The linearity was good (r ² > 0.999), with intra‐ and interday precision levels of 0.43–7.87% and 0.65–9.95% and the intra‐ and interday accuracies of 84.36–115.82% and 83.78–118.69%, respectively. The stability (relative standard deviation) was <14.75%. The mean recovery was 80.81–117.86% (relative standard deviation <10.00%). Ninety‐seven commercial dietary supplements available in South Korea were analyzed. While none contained detectable amounts of the 35 antihypertensive compounds, the developed LC/MS/MS procedure can be used for routine analysis to monitor illegal adulteration in various forms of dietary supplements.     

Fast quantitative determination of methylphenidate levels in rat plasma and brain ex vivo by MALDI‐MS/MS

This study presents a simple and sensitive high‐throughput matrix‐assisted laser desorption/ionization time‐of‐flight tandem mass spectrometry (MALDI‐MS/MS) method for ex vivo quantification of methylphenidate (MPH) in rat plasma and brain. The common MALDI matrix alpha‐cyano‐4‐hydroxycinnamic acid was used to obtain an optimal dried droplet preparation. For method validation, standards diluted in plasma and brain homogenate prepared from untreated (control) rats were used. MPH was quantified within a concentration range of 0.1–40 ng/ml in plasma and 0.4–40 ng/ml in brain homogenate with an excellent linearity (R² ≥ 0.9997) and good precision. The intra‐day and inter‐day accuracies fulfilled the FDA's ±15/20 critera. The recovery of MPH ranged from 93.8 to 98.5% and 87.2 to 99.8% in plasma and homogenate, respectively. We show that MPH is successfully quantified in plasma and brain homogenate of rats pre‐treated with this drug using the internal standard calibration method. By means of this method, a linear correlation between plasma and brain concentration of MPH in rodents pre‐treated with MPH was detected. The simple sample preparation based on liquid‐liquid extraction and MALDI‐MS/MS measurement requires approximately 10 s per sample, and this significantly reduces analysis time compared with other analytical methods. To the best of our knowledge, this is the first MALDI‐MS/MS method for quantification of MPH in rat plasma and brain. Copyright © 2015 John Wiley & Sons, Ltd.     

Bioanalytical assay development and validation for simultaneous quantification of five schisandra lignans in rat primary hepatocytes based on LC‐MS/MS: application to a real‐time uptake study for Schisandra Lignan Extract

Schisandra lignans, mainly including schizandrol A, schizandrol B, schisantherin A, schizandrin A, schizandrin B, etc., are the major active ingredients of Schisandra chinensis . In the present study, a robust liquid chromatography–tandem mass spectrometric (LC‐MS/MS) method was developed for the simultaneous quantification of schisandra lignans in rat primary hepatocytes. Lovastatin was used as an internal standard, and chromatographic separation was achieved on a Shimadzu C₁₈ column with a gradient elution at the flow rate of 0.2 mL/min. All of the analytes were detected in multiple reaction monitoring mode with positive electrospray ionization since the sodium adduct ion [M + Na]⁺ was observed as the most intensive peak in the MS spectrum. For schizandrol A, schisantherin A and schizandrin A, the dynamic range was within 2–1000 ng/mg protein, and the linear range of schizandrol B and schizandrin B was from 5 to 1000 ng/mg protein. The intra‐ and inter‐day precision was <15% and the accuracy (relative error) ranged from −15 to 15%. No significant variation was observed in the stability tests. The validated method was then successfully applied to the time‐dependent uptake study for the Schisandra Lignan Extract in rat primary hepatocytes.