Analysis of omnoponum by surface-ionization mass spectrometry and liquid chromatography tandem mass spectrometry methods

This paper provides the development of analytical capabilities of surface-ionization mass spectrometry (SI/MS) and high performance liquid chromatography with tandem mass spectrometry (HPLC/MS/MS) for narcotic analgesic omnoponum, which perfectly exemplifies a mixture of opium alkaloids. It has been revealed that the investigated opiates solution, omnoponum, is ionized by the surface ionization (SI) method with high sensitivity. In the SI mass spectrum, M⁺, (M−H)⁺, (M−H−2nH)⁺, (M−R)⁺ and (M−R−2nH)⁺ ion lines, where M is a molecule, H is the hydrogen atom and R is a radical, were observed. These ion lines consist of combined omnoponum mixture SI mass spectra, i.e. morphine, codeine, thebaine, papaverine, and narcotine. Moreover, while the study of omnoponum by HPLC/MS/MS methods has attested that the mixture really consists of 5 components, it has been demonstrated that the SI/MS method can be utilized for the analysis of this mixture without the necessity of its chromatographic separation.     

Quantification of artemisinin in human plasma using liquid chromatography coupled to tandem mass spectrometry

A liquid chromatographic tandem mass spectroscopy method for the quantification of artemisinin in human heparinised plasma has been developed and validated. The method uses Oasis HLB™ μ-elution solid phase extraction 96-well plates to facilitate a high throughput of 192 samples a day. Artesunate (internal standard) in a plasma–water solution was added to plasma (50 μL) before solid phase extraction. Artemisinin and its internal standard artesunate were analysed by liquid chromatography and MS/MS detection on a Hypersil Gold C18 (100 mm × 2.1 mm, 5 μm) column using a mobile phase containing acetonitrile–ammonium acetate 10 mM pH 3.5 (50:50, v/v) at a flow rate of 0.5 mL/min. The method has been validated according to published FDA guidelines and showed excellent performance. The within-day, between-day and total precisions expressed as R.S.D., were lower than 8% at all tested quality control levels including the upper and lower limit of quantification. The limit of detection was 0.257 ng/mL for artemisinin and the calibration range was 1.03–762 ng/mL using 50 μL plasma. The method was free from matrix effects as demonstrated both graphically and quantitatively.     

Simple and accurate quantitative analysis of ten antiepileptic drugs in human plasma by liquid chromatography/tandem mass spectrometry

A simple, accurate, and sensitive liquid chromatography (LC)-tandem mass spectrometry (MS/MS) method has been developed for the simultaneous quantification of 10 antiepileptic drugs (AEDs; gabapentin (GBP), levetiracetam (LEV), valproic acid (VPA), lamotrigine (LTG), carbamazepine-10,11-epoxide (CBZ-epoxide), zonisamide (ZNS), oxcarbazepine (OXC), topiramate (TPM), carbamazepine (CBZ), phenytoin (PHT)) in human plasma as a tool for drug monitoring. d₁₀-Phenytoin (d₁₀-PHT) and d_6-valproic acid (d₆-VPA) were used as internal standards for the positive- and negative-ionization modes, respectively. Plasma samples were precipitated by the addition of acetonitrile, and supernatants were analyzed on a C18 reverse-phase column using an isocratic elution. Detection was carried out in selected reaction monitoring (SRM) mode. The calibration curves were linear over a 50-fold concentration range, with correlation coefficients (r²) greater than 0.997 for all AEDs. The intra- and inter-day precision was less than 12%, and the accuracy was between 85.9 and 114.5%. This method was successfully used in the identification and quantitation of AEDs in patients undergoing mono- or polytherapy for epilepsy.     

Identification of gentamicin impurities by liquid chromatography tandem mass spectrometry

An HPLC/MS/MS method was developed for identification of impurities in gentamicin. The HPLC was performed on a Synergy Hydro-RP column using 50 mM trifluoroacetic acid (TFA), pH 2 adjusted with ammonium solution and methanol as mobile phase. All impurities in gentamicin were separated from main gentamicin components. Atmospheric pressure chemical ionization (APCI) was used and product mass spectra of protonated molecules were acquired. Seventeen impurities were detected in gentamicin. Reference compounds: gentamicins: C_2b, B, B₁, G-418, sisomicin, garamine and gentamines: C₁, C_1a, C₂, C_2a were used for spectra interpretation and impurities identification. All MS/MS spectra were interpreted and fragmentation transitions for gentamicins and in general for aminoglycoside antibiotics (AG) were proposed. All impurities were identified. More than one isomere were proposed for three impurities.     

A liquid chromatography/tandem mass spectrometry assay to quantitate MS-275 in human plasma

A rapid, sensitive and selective method was developed and validated using LC/MS/MS for determination of MS-275 in human plasma. Sample preparation involved a single step liquid-liquid extraction by the addition of 0.2 ml of plasma with 5 ml acetonitrile/n-butyl-chloride. Separation of the compounds of interest, including the internal standard paclitaxel, was achieved on a Waters X-Terra C(18) (50 mm x 2.1mm i.d., 3.5 microm) analytical column using a mobile phase consisting of acetonitrile/ammonium acetate (pH 2.9; 2mM)(60:40, v/v) containing 0.1% formic acid and isocratic flow at 0.15 ml/min for 3 min. The analytes were monitored by tandem-mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 0.5-100 ng/ml with values for the coefficient of determination of >0.99. The values for both within day and between day precision and accuracy were well within the generally accepted criteria for analytical methods (<15%). This method was subsequently used to measure concentrations of MS-275 in cancer patients receiving an oral weekly dose of 4 mg/m(2).     

Identification and determination of nucleosides in rat brain microdialysates by liquid chromatography/electrospray tandem mass spectrometry

A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed for the determination of brain basal nucleosides (inosine, guanosine and adenosine) in microdialysates from the striatum and cortex of freely moving rats. A microdialysis probe was surgically implanted into the striatum or cortex of individual rats and Ringer's solution was used as the perfusion medium at a flow rate of 0.3 or 0.5 μl/min. The samples were then analyzed off-line by LC/MS/MS experiments. The separation of inosine, guanosine and adenosine was carried out on a cyano column using a mobile phase of 10 mM ammonium acetate, 1% acetic acid and 8% methanol at a flow rate of 0.4 ml/min. Analytes were detected by electrospray ionization tandem mass spectrometry in the positive ion mode. The detection limit for inosine, guanosine and adenosine was 80, 80 and 40 pg on column, respectively. With this method, the intercellular basal inosine, guanosine and adenosine concentrations in striatum and cortex of rat were determined.     

PMEA-Na在beagle犬血浆中的液相色谱/质谱/质谱联用法测定及其药代动力学

目的建立LC/MS/MS法测定犬血浆中PMEA-Na浓度,进行其药代动力学研究.方法血浆样品经甲醇沉淀蛋白后,采用多反应监测法测定其血药浓度.色谱柱为Xterra MS柱,流动相为甲醇水甲酸(25750.5),流速为 0.25 ml·min-1.Beagle犬分3个剂量组经静脉给药,给药剂量分别为 1.0、3.0 和 6.0 mg·kg-1.药代动力学参数通过DAS软件计算获得.结果PMEA-Na线性范围0.02～20 mg·L-1 (r=0.999);最低检测浓度为 20 μg·L-1,方法回收率为 97.1%～107.3%,日内日间变异分别小于 6.5%、10.8%.beagle 犬在 1.0, 3.0 与 6.0 mg·kg-1剂量下单次iv PMEA-Na后,测得其AUC分别为 2.3±0.5, 8.2±1.3 and 18.5±1.3 mg·L-1·h; t1/2 为 3.9±1.8, 8.4±1.5 and 8.9±0.6 h; CL为 0.44±0.09, 0.35±0.05 and 0.31±0.03 ml·h-1·kg-1.结论本方法专属性强,准确性好,可用于PMEA-Na血药浓度测定和药代动力学研究.     

Comprehensive chemical analysis of Venenum Bufonis by using liquid chromatography/electrospray ionization tandem mass spectrometry

A rapid, sensitive and versatile liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method was developed for the comprehensive analyses of the chemical constituents contained in the Chinese medicine-Venenum Bufonis (VB, Chan’ Su in Chinese). LC analysis was carried out on an Agilent Eclipse plus C₁₈ RRHD column (2.1 × 150 mm, 1.8 μm) with a linear gradient solvent system of water (0.1% formic acid) and acetonitrile (0.1% formic acid) as mobile phase. Detection and quantification were performed by multiple reaction monitoring (MRM) transitions via electrospray ionization (ESI) source operating in the positive ionization mode. Through “Molecular Feature Extraction” (MFE), more than 900 features were detected from VB extracts. Among them, a total of 97 components were identified using the Agilent METLIN accurate mass matching database (DB) established according to those reported in the literatures. Further more, 30 high quality matches were obtained by comparisons of their accurate mass and retention times (AMRT) with those imported out in the developed personal database (METLIN DB with AMRT). The characteristic fragmentation pathways were proposed for the tentative characterization of four representative types of bufadienolides in the present work. The targeted MS/MS experiment of the 30 major compounds was performed for their quantification and semi-quantification. And 7 of them were quantified over the assaying concentration range of 5.0-500 pg/μL. The lowest limit of detection and quantification of them were 0.25-0.50 and 1.25-0.25 pg/μL, respectively. The recoveries varied from 83 to 106% depending on the chemical types and different extraction solvents. The remained 23 bufosteroids were simultaneously semi-quantified using three representative standard compounds as their standard references, respectively.     

Simultaneous determination of lithospermic acid B and its three metabolites by liquid chromatography/tandem mass spectrometry

A rapid and sensitive method based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed for the simultaneous determination of lithospermic acid B and its three main O-methylated metabolites in rat serum with silibinin as the internal standard. The calibration curves for LSB, and the three metabolites were linear over the ranges of 16–4096, and 8–2048 ng/ml, respectively, with coefficients of correlation >0.998. For LSB, the intra-assay coefficient of variance (CV) was less than 9.3% and the inter-assay CV was less than 8.9%. The inter-assay mean accuracy was between 92.8% and 104.7%. For the three metabolites, the intra-assay CV was less than 8.7% and the inter-assay CV was less than 9.9%. The inter-assay mean accuracy was between 92.5% and 107.9%. This quantitation method was successfully applied to a pharmacokinetic study of LSB in rats. Also, a total recovery of 5.2%was found in bile after oral administration.     

Detection of 10 sweeteners in various foods by liquid chromatography/tandem mass spectrometry

The analytical method for sweeteners in various food matrixes is very important for food quality control and regulation enforcement. A simple and rapid method for the simultaneous determination of 10 sweeteners [acesulfame potassium (ACS-K), aspartame (ASP), cyclamate (CYC), dulcin (DUL), glycyrrhizic acid (GA), neotame (NEO), neohesperidin dihydrochalcone (NHDC), saccharin (SAC), sucralose (SCL), and stevioside (STV)] in various foods by liquid chromatography/tandem mass chromatography (LC–MS/MS) was developed. The chromatographic separation was performed on a Phenomenex Luna Phenyl-Hexyl (5 μm, 4.6 mm × 150 mm) column with gradient elution of 10 mM ammonium acetate in water and 10 mM ammonium acetate in methanol. The recoveries of the 10 sweeteners were between 75% and 120%, and the coefficients of variation were less than 20%. The limits of quantification were 0.5 μg/kg for NHDC and SCL. For the other sweeteners, the limits of quantification were 0.1 μg/kg. Compared to the traditional high-performance liquid chromatography method, the LC–MS/MS method could provide better sensitivity, higher throughput, enhanced specificity, and more sweeteners analyzed in a single run. The samples included 27 beverages (16 alcoholic and 11 nonalcoholic beverages) and 15 pickled foods (1 pickled pepper, 3 candies, and 11 candied fruits). Two remanufactured wines were found to contain 7.2, 8.5 μg/g SAC and 126.5, 123 μg/g CYC, respectively. ACS-K, ASP, SCL, and NEO were detected in five beverages and drinks. The pickled peppers and candied fruits were found to contain SAC, GA, CYC, ASP, STV, NEO, and ACS-K. The wine with sweeteners detected was remanufactured wine, not naturally fermented wine. Therefore, the ingredient label for the sweeteners of remanufactured wine should be regulated by the proper authority for inspection of sweeteners.     

Liquid chromatography/tandem mass spectrometry method for determination of olanzapine and N-desmethylolanzapine in human serum and cerebrospinal fluid

A validated, accurate and sensitive LC–MS/MS method for determination of olanzapine and its metabolite N-desmethylolanzapine has been developed. The analytes were quantified by tandem mass spectrometry operating in positive electrospray ionization mode with multiple reaction monitoring. Olanzapine and desmethylolanzapine were extracted from serum or cerebral spinal fluid samples, 200 μl, with tert-butyl methyl ether using olanzapine-D3 as internal standard. Calibrations for olanzapine and desmethylolanzapine were linear within the selected range of 0.2–30 ng/ml (6–96 nM) in cerebral spinal fluid and for olanzapine in plasma, in the range of 5–100 ng/ml (16–320 nM). The method was successfully used for the analysis of samples from patients treated with olanzapine in the dose range of 2.5–25 mg/day.     

Liquid chromatography/tandem mass spectrometry utilizing ion-molecule reactions and collision-activated dissociation for the identification of N-oxide drug metabolites

A liquid chromatography/tandem mass spectrometry (LC/MS³) method based on ion-molecule reactions and collision-activated dissociation (CAD) is presented for the identification of analytes with the N-oxide functional group directly in mixtures. Tri(dimethylamino)borane (TDMAB) rapidly and selectively derivatizes protonated N-oxides in a modified commercial linear quadrupole ion trap (LQIT) mass spectrometer to yield a distinct product ion (adduct—(CH₃)₂NH). The LQIT was outfitted with an external reagent-mixing manifold that allows TDMAB to be mixed with the helium buffer gas used in the trap. The derivatized analytes are readily identified on the basis of a shift of 98 Th (Thomson) relative to the m/z value of the protonated analyte. Further probing of the derivatized analytes via isolation followed by CAD can be used to confirm the presence of an N-oxide, and distinguish between aliphatic and aromatic tertiary N-oxides. Since the ion-molecule reaction is fast, these experiments can be accomplished on the same time scale as typical CAD-based MSⁿ experiments, thus maintaining the duty cycle of the instrument for this type of experiment. To demonstrate real world applicability, the method was tested on real active pharmaceutical ingredients and their derivatives.     

液-质联用法研究磷霉素钙在健康人体的生物等效性

目的:建立液-质联用法测定人血浆中磷霉素钙的浓度,研究磷霉素钙的人体药动学及生物等效性。方法:血浆样品中加入内标阿德福韦,经乙腈蛋白沉淀,SB-Aq C18色谱柱进行分离,流动相流速为1.0mL.min-1,梯度洗脱方式,采用多反应监测(MRM)的负离子方式检测。结果:在100~25 000μg.L-1范围内呈良好的线性关系,日内及日间精密度(RSD)小于8%。受试制剂和参比制剂的tmax分别为(1.8±0.2)h和(2.4±0.5)h,Cmax分别为(6 034.0±1 540.9)μg.L-1和(6 182.5±1 221.4)μg.L-1,AUC0-t分别为(43 755.6±7 198.0)μg.h.L-1和(43 720.1±9 134.2)μg.h.L-1。结论:本法准确,灵敏无杂质干扰。两种制剂生物等效。     

Urine thebaine determination by liquid chromatography tandem mass spectrometry after poppy seed consumption

Laboratories are challenged to distinguish whether a positive urine morphine result is due to heroin use or possible poppy seed consumption. Thebaine is an opium alkaloid that has been shown to be present in the urine of individuals who have consumed poppy seeds, as well as those who have used opium. It is not present in heroin. We present a sensitive, specific liquid chromatography tandem mass spectrometry (LC–MS/MS) assay for thebaine. We show that thebaine is detectable after consumption of two different poppy seed-containing products for up to 72 h in urine. We discuss limitations of the assay and suggest how the test might best be used.     

Identification of etamiphylline and metabolites in equine plasma and urine by accurate mass and liquid chromatography/tandem mass spectrometry

Etamiphylline camsylate (Millophylline V) was administered intravenously to two horses at a dose of 2.8 mg/kg. Urine and blood samples were taken up to 32 h post administration. Unhydrolyzed plasma and urine was extracted using solid phase extraction (SPE). The identity of the parent drug and metabolites was confirmed using a linear ion trap mass spectrometer and accurate mass analysis on an orbitrap mass spectrometer. Desethyletamiphylline (molecular weight 251) was the main metabolite observed in the urine and plasma samples and resulted from the N‐deethylation of etamiphylline. The second metabolite detected in urine and plasma resulted from the demethylation of etamiphylline (molecular weight 265). The third minor metabolite detected in urine was proposed to have resulted from a simultaneous N‐deethylation and demethylation of etamiphylline (molecular weight 238). Copyright © 2010 John Wiley & Sons, Ltd.     

Analysis of deoxyribonucleotide pools in human cancer cell lines using a liquid chromatography coupled with tandem mass spectrometry technique

Endogenous ribonucleotides and deoxyribonucleotides play a critical role in cell function, and determination of their levels is of fundamental importance in understanding key cellular processes involved in energy metabolism and molecular and biochemical signaling pathways. In this study, we determined the respective ribonucleotide and deoxyribonucleotide pool sizes in different human cell lines using a simple sample preparation method and LC/MS/MS. This assay was used to determine alterations in deoxyribonucleotide pools in human pancreatic PANC1 cells in response to hypoxia and to treatment with either hydroxyurea or aphidicolin. The levels of all deoxyribonucleotide metabolites decreased with hypoxia treatment, except for dUMP, which increased by two-fold. This LC/MS/MS assay is simple, fast, and sensitive, and it represents a significant advance over previously published methodologies.     

In vivo saturation binding of GABA‐A receptor ligands to estimate receptor occupancy using liquid chromatography/tandem mass spectrometry

Typically, the dose‐occupancy curves for GABA‐A receptor ligands are determined using in vivo binding of [³H]flumazenil. This study describes in vivo binding experiments without the use of tracer ligands. Bound and free fractions were measured directly using a highly sensitive LC/MS/MS detection method after in vivo administration of the GABA‐A ligands zolpidem, (RS)‐zopiclone, L‐838417 and flumazenil, to demonstrate affinity and saturation of the filter‐retained, membrane‐bound fraction. The in vivo binding of flumazenil and L‐838417 both saturated around 200 nm , at a similar level to the specific binding of (S)‐zopiclone after doses of the racemic zopiclone, using (R)‐zopiclone to estimate non‐specific binding. This saturable component represented an estimate of benzodiazepine binding sites available on GABA‐A receptors in vivo (200 nm ). Dose‐occupancy curves were constructed to estimate the dose required to achieve 50% occupancy and matched estimates obtained with tracer methods. In contrast to tracer methods, this method is uniquely suitable to the demonstration of stereoselective binding of the (S)‐isomer in vivo after doses of racemic zopiclone. These results demonstrate that the LC/MS/MS measurements of total drug concentrations typically used in early drug development can be adapted to provide information about receptor occupancy in vivo. Copyright © 2009 John Wiley & Sons, Ltd.     

Analysis of iridoid glucosides in Hedyotis diffusa by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

An HPLC-DAD-ESI-MS/MS method was developed for analysis of iridoid glucosides (IGs) from Hedyotis diffusa Willd. The optimized separation condition was achieved with the Complex Sample Analysis Software System (CSASS) software, under which the whole analytes were achieved complete resolution especially for some isomeric IGs. Based on the UV and fragmentations, eleven IGs were detected. According to the fragmentation patterns of the three standard IGs, especially those of the isomeric standards, seven IGs including three pairs of isomers were unambiguous/tentatively identified. For the isomeric IGs with methyl ester or carboxyl group at C-4, the extents of the losses of CH(3)OH and/or H(2)O from their molecular and/or the aglycone adducts are useful for the differentiation of the stereoisomers in positive ion (PI) mode, which depends on the stereochemistry of the hydroxyl group on the cyclopentanoid unit.     

Quantitation of tetrahydrocannabinol in hair using immunoassay and liquid chromatography with tandem mass spectrometric detection

A quantitative analytical procedure for the determination of Δ⁹‐tetrahydrocannabinol (THC) in hair has been developed and validated using liquid chromatography with tandem mass spectral detection (LC‐MS/MS). Specimens that were determined as containing cannabinoids following immunoassay testing were quantified using solid‐phase extraction followed by liquid chromatographic separation and tandem mass spectral detection in positive electrospray ionization mode. For confirmation, two transitions were monitored and one ratio determined. Samples being reported as positive were required to have both transitions present, the ratio of quantifying transition to qualifying transition being within 20% of that determined from known calibration standards. The limit of quantitation and the limit of detection was 10 pg/mg. The percentage recovery of the THC from hair at 20 pg/mg was 56% and a matrix effect of the hair showed an ion suppression percentage of ?51%. The immunochemical screening method was performed following a rapid aqueous extraction, requiring only 10 mg of hair; the confirmatory procedure required 20 mg of hair. The methods were applied to proficiency specimens from the Society of Hair Testing, which had been received in August 2008. Copyright © 2009 John Wiley & Sons, Ltd.     

Development and validation of liquid chromatography tandem mass spectrometry methods for the determination of gentamicin, lincomycin, and spectinomycin in the presence of their impurities in pharmaceutical formulations

Liquid chromatography with tandem mass spectrometry (LC/MS/MS) methods for the determination of gentamicin, lincomycin and spectinomycin in the presence of their impurities were developed and tested. Chromatographic separations were achieved using gradient elution on a C18 column. All components were ionized by positive-ion electrospray and detected by multi reaction monitoring (MRM) with an LC-tandem mass spectrometer. Calibration curves were linear with correlation coefficients better than 0.99. The developed method for the determination of gentamicin provides complete base line separation of components C1, C1a, C2, C2a and C2b mentioned in the European and British Pharmacopoeias. The second developed method makes possible a simultaneous analysis of the active compounds of both lincomycin and spectinomycin. Additionally, all impurities defined in the pharmacopoeias for all three active components were determined and their identities confirmed. The methods were tested in routine quality control analysis.