A method for the low-level (ng g(-1)) determination of perfluorooctanoate in paper and textile by liquid chromatography with tandem mass spectrometry

The determination of perfluorooctanoate (PFO) in articles of commerce has become increasingly important to understand if treated products are a possible source of PFO. An LC-MS/MS method for the determination of PFO in paper and textile using a dual labeled 13C-PFOA internal standard was successfully developed and validated. Residues of PFO were determined using an isocratic, reversed-phase high-performance liquid chromatography (HPLC) method with an ammonium acetate/methanol buffer. Ions monitored were 413 (parent) and 369 (daughter) for PFO and 415 (parent) and 370 (daughter) for dual labeled 13C-PFOA internal standard. As a precaution against ubiquitous PFO that occasionally occurs in mobile phase or instrument components, two Hypercarb cartridges (4 mm) were placed before the HPLC injector. Any PFO that was captured by the cartridges was removed before each injection by flushing the system with 100% methanol prior to equilibration with the isocratic mobile phase. Overall recovery and standard deviation over a 3 day validation regimen for samples (n=54-55) fortified with PFOA at 5, 50, and 200 ng g(-1) were 114+/-4.9% for textile and 110+/-7.6% for paper. The results also established a limit of detection (LOD) of 1 ng g(-1) in textile and 2 ng g(-1) in paper based upon S/N of the 5.0 ng g(-1) fortification versus the untreated paper and textile.     

Determination of extractable perfluorooctanoic acid (PFOA) in water, sweat simulant, saliva simulant, and methanol from textile and carpet samples by LC/MS/MS

Methods were developed to quantify the amount of perfluorooctanoic acid (PFOA) extracted from textile and carpet samples through contact with water, methanol, and sweat and saliva simulants using LC/MS/MS. The limit of quantitation (LOQ) for samples extracted in water and sweat simulant is 1 ppb (ng PFOA (g sample)(-1)) while the limits of quantitation for samples extracted in saliva simulant and methanol were 3 ppb and 2.5 ppb, respectively. Method validation results are provided for a polyester control textile sample that was extracted in water on two different days by different analysts, which gave an overall recovery of 103% and standard deviation of 5.3% for 30 analyses. However, for routine application of these methods to a large number of sample sets differing in chemical and physical compositions, a complete validation for each sample type is not practical or possible since control samples for fortifications are often not available. Instead, suitable analytical methods and acceptance criteria are described which ensure accurate PFOA quantitation in each of the solvent extract types. During routine use of these methods, post-extraction spike recoveries for the different sample types and solvents are 100 +/- 15% using a dual isotopically labeled (13)C-PFOA internal standard to correct for matrix effects. A comparison of extraction solvent versus time using a wrist action shaker for textile and carpet samples demonstrates that the total extractable amount of PFOA is similar for each of the solvent types. However, as expected the rate of extraction in water and simulants is significantly less than that of methanol. Finally, a comparison of 2 h and 24 h wrist action shaker extractions with a 1.5 h pressurized fluid extraction (PFE) in methanol reveals that the 24 h wrist action shaker yields the highest results. The 2 h wrist action shaker results are similar to those of the 1.5 h PFE extraction.     

超高效液相色谱-三重四极杆质谱联用检测红色糖多孢菌胞内中间代谢物同位素丰度

采用超高效液相色谱-三重四极杆串联质谱联用仪(LC-MS/MS),建立了能精确检测红色糖多孢菌胞内代谢物13C同位素丰度的方法.在优化的超高效液相色谱的条件及三重四极杆串联质谱的离子传输电压和碰撞池电压下,筛选出各胞内代谢物的最佳离子对.根据物质在LC-MS/MS中生成的母离子和子离子碳链长短及子离子是否含有13C等特性,建立了"一对一"法、"一对多"法和单级质谱SIM法等同位素丰度检测方法.利用这些方法,检测了自然标记标准品和13C标记实验样品,根据实验值与理论值的接近程度筛选出最优方法.结果表明,对于以磷酸糖类为代表的子离子不含有13C的代谢物,"一对一"法最有效;对于以有机酸类为代表的母离子和子离子都含有13C的短碳链代谢物,"一对多"法更有优势;对于以辅酶A类为代表的母离子和子离子都含有13C且碳链较长的代谢物,单级质谱SIM法才能发挥作用.建立的同位素丰度检测方法具有较好的准确度,可应用于红色糖多孢菌胞内代谢物同位素丰度的检测,为后续研究菌体的代谢机理,实现红霉素的高效表达奠定了基础.     

Sensitive and rapid simultaneous quantitation of leucovorin and its major active metabolite 5-methyl-tetrahydrofolate in human plasma using a liquid chromatography coupled with triple quadruple mass spectrometry

Bioanalysis of an endogenous compound such as leucovorin is never an easy task on a liquid chromatography tandem mass spectrometer (LC–MSMS). Unless it is necessary, regulatory guidance discourages working with surrogate matrices for calibration curve standard preparation. Herein, a selective and sensitive liquid chromatography–tandem mass spectrometry method for simultaneous determination of leucovorin and 5-methyl tetrahydrofolic acid in human plasma was developed and validated. Stable labeled internal standards, i.e. leucovorin D₄ and 5- methyl tetrahydrofolic acid ¹³C₅, were used as internal standards to track and compensate the parent compounds during processing and extraction from plasma. The method involves a rapid solid-phase extraction from plasma followed by reverse-phase gradient chromatography and mass spectrometry detection with a total run time of 5 min. The method was developed and validated from 5 to 2,202 ng/ml for leucovorin and from 5 to 1,300 ng/ml for 5-methyl tetrahydrofolic acid. The mean recoveries for leucovorin and 5-methyl tetrahydrofolic acid were 100.4 and 100.9% respectively. The validated method enabled the simultaneous analysis of leucovorin and 5-methyl tetrahydrofolic acid in samples from clinical pharmacokinetic studies of leucovorin. The peak concentrations of leucovorin and 5-methyl tetrahydrofolic acid were 651–883 and 518–635 ng/ml, respectively, in fasted and fed conditions. The terminal half-life values for leucovorin and 5-methyl tetrahydrofolic acid were 9.3–10.5 and 9.2–17.6 h, respectively.     

海河底泥中12种抗生素残留的液相色谱串联质谱同时检测

建立了同时检测河流底泥中12种抗生素(4种磺胺类、3种喹诺酮类、2种四环素类、2种大环内酯类、甲氧苄啶)残留的高效液相色谱串联质谱检测方法(HPLC-MS/MS)。样品提取过程中以NaF为离子交换剂,经2种提取液逐次提取,合并提取液,正己烷脱脂,萃取物经SAX-HLB固相萃取系统净化浓缩,氮吹,定容。以乙腈和0.3%甲酸为流动相,采用梯度洗脱进行液相色谱分离,以Simatone为内标物,用质谱检测器进行定性和定量分析。12种目标物的检出限为1.0~5.0 ng/g,回收率为65%~91%,相对标准偏差为0.6%~5.1%(n=4)。     

Novel Dual Two‐Dimensional Liquid Chromatography Online Coupled to Ultraviolet Detector,Fluorescence Detector,Ion‐Trap Mass Spectrometer for Short Peptide Amino Acid Sequence Determination with Bottom‐Up Strategy

A novel dual two‐dimensional (2D) high‐performance liquid chromatography (LC) setup coupled online to an ultraviolet (UV) detector, fluorescence (FL) detector, and ion‐trap mass spectrometer (MS) has been developed for determining the amino acid sequence of short peptides using a novel bottom‐up strategy. Short peptides were electrothermally hydrolyzed to shorter peptides and amino acid enantiomers. The first 2D LC‐UV and FL system was used to separate and identify the produced parent and daughter short peptides and amino acid isomers and enantiomers in the hydrolysate; the second 2D LC‐MS was used to identify the presence of cysteine and obtain the molecular mass signals and N‐terminal peptide fragment ion signals for parent and daughter short peptides. The identified amino acid enantiomers are used to form any possible short peptides by permutation and combination in an order from dipeptide to a tripeptide, to a tetrapeptide, and to even higher short peptides. The correct short peptides are confirmed by comparing the molecular weights of the constituent amino acid enantiomers and the molecular weights of identified short peptides together, with the characteristic N‐terminal peptide fragment ion signals. The amino acid sequence of the dipeptide ester aspartame and the tripeptide glutathione was successfully determined by this method.     

高效液相色谱-串联质谱法测定食品中曲酸

建立了食品中新型防腐剂曲酸的高效液相色谱-串联质谱的定量测定方法。动物禽肉、鱼虾甲壳类、酱菜类、水果蔬菜、面制品等固体样品经乙腈提取;酱及酱油、醋、酒、饮料、糖浆等液体样品经水稀释,乙酸锌和亚铁氰化钾沉淀蛋白;以C18柱为分离柱,流动相为乙腈和5 mmol/L乙酸铵甲酸溶液,采用电喷雾串联四极杆质谱进行检测。选择1个母离子和2个子离子进行选择反应监测,以13C6-曲酸作为内标,选择信号最强的子离子进行定量测定。固体类基质中的定量限(按信噪比(S/N)大于10计)为0.1 mg/kg;液体类基质中的定量限为2.5 mg/kg。在0.1～2.0 mg/L范围内呈现良好的线性关系,相关系数r＞0.99。各种基质在3个添加水平的平均回收率在72.6%～114%之间,相对标准偏差均小于11.4%。本方法简单实用,准确可靠,适用范围包括了食品中可能使用曲酸这种食品添加剂的大部分基质,可以满足进出口食品中曲酸的定性和定量要求。     

Rapid and simultaneous determination of hexapeptides (Ac-EEMQRR-amide and H2N-EEMQRR-amide) in anti-wrinkle cosmetics by hydrophilic interaction liquid chromatography-solid phase extraction preparation and hydrophilic interaction liquid chromatography with tandem mass spectrometry

A rapid method for the simultaneous determination of Ac-EEMQRR-amide and H(2)N-EEMQRR-amide in cosmetic products was developed and evaluated. This analytical procedure involved extracting samples with 0.1:0.1:85:15 (v:v) trifluoroacetic acid (TFA):formic acid:acetonitrile (ACN):water and determination by hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC-MS/MS). Samples showing serious ion suppression were further cleaned up using HILIC-SPE prior to HILIC-MS/MS analysis. Stable isotopically labeled peptides, corresponding to the above two peptides, were used as internal standards to correct for loss of recovery and matrix effects. Electrospray ionization (ESI) in the positive mode was used. The linear range was 2.0-1000 ng/mL for Ac-EEMQRR-amide and 25.0-2500 ng/mL for H(2)N-EEMQRR-amide. Thirteen commercial products were analyzed for the two peptides using this method. The amounts of Ac-EEMQRR-amide in the samples ranged from none detected to 42.3 μg/g. H(2)N-EEMQRR-amide was not detected in any of the samples. The recoveries for Ac-EEMQRR-amide and H(2)N-EEMQRR-amide ranged from 85% to 110% and 84% to 119%, respectively, at the spiking level of 30 μg/g.     

Determination of the tricyclic compound adosupine and its three metabolites in plasma and brain of rat using high-performance liquid chromatography.

An analytical method for the detection in biological samples of the novel tricyclic compound adosupine (10-acetoamido-5-methyl-5,6-dihydro-11H-dibenzo[b,e]azepin-6 ,11-dione), which is capable of influencing various forms of urinary bladder hyperreflexia has been developed using high-performance liquid chromatography with UV detection. Liquid-liquid extraction was used to isolate the parent compound, three metabolites and an analogue (added as internal standard) from plasma and brain of rat. Adosupine was well separated from its three metabolites with 0.01 M disodium hydrogenphosphate-acetonitrile-methanol-nonylamine (59.986:38:2:0.014) at pH 4.5 as mobile phase using a C18 reversed-phase column. The standard curves were linear in the range 50-5000 ng/ml (or ng/g) for adosupine and metabolites in both plasma and brain. The between- and within-assay variations for high and low concentrations of the parent compound and the three metabolites were 8.2-14%. In the range 50-5000 ng/ml (or ng/g) the accuracy of the method was satisfactory, with the relative error always lower than 10%. Analytical recoveries of added adosupine and the three metabolites were higher than 82%. The method has been applied successfully, to investigate the pharmacokinetics of the drug and its distribution in the central nervous system of rats.     

Determination of terbinafine in tissues

Terbinafine and N-demethyl terbinafine concentrations were determined simultaneously in rat tissues by a high-performance liquid chromatography method. This method involved the homogenization of tissues (except for skin) followed by a liquid-liquid extraction. Skin samples were dissolved in sodium hydroxide prior to extraction. Terbinafine and its N-demethylated metabolite were assayed using a C(18) reversed-phase column with a mobile phase of acetonitrile and water (40:60) containing ortho phosphoric acid (0.02 M) and triethylamine (0.01 M), and UV detection (at 224 nm). The standard curve for the assay (constructed using clotrimazole as internal standard) was linear over the concentration range 100-3000 ng/g in skin and 10-600 ng/g in all other tissues. The inter- and intra-day precision for both terbinafine and metabolite was between 0.2% and 16%. The limit of quantification was 10 ng/g in all tissues and 100 ng/g in skin. This assay was found to be reliable and reproducible for the determination of terbinafine and N-demethyl terbinafine concentration in all rat tissues and has been used for tissue distribution studies.     

小硅藻光合作用脂13 C稳定同位素定量标记分析

以小硅藻( Nitzschia closterium f. minutissima)作为研究对象,利用超高压液相色谱联用四极杆-静电场轨道阱高分辨质谱技术,研究了处于平台期的小硅藻中的光合作用脂被13 C标记的程度和速度。结果显示：在平台期13 C人工标记的10天内,所有4类光合作用脂均被13 C明显标记,其中二酰甘油双半乳糖脂( DG-DG)、磷脂酰甘油( PG)、二酰甘油硫代糖脂( SQDG)被13 C标记的总量,从刚进入平台期到平台期第8天逐渐增加,此后出现下降趋势,在第8天分别达到最大值(173±24) ng/mg,(473±41) ng/mg 及(1224±21) ng/mg,标记率分别达到56.3%,38.4%和62.6%;而二酰甘油单半乳糖脂(MGDG)被13C标记的总量在整个平台期呈现持续上升趋势,并在第10天达到(956±51) ng/mg,标记率为87.3%。可见,不同脂质在藻体内合成的先后时间有差别,为了清晰地了解生物摄食过程中对微藻中特定脂类的同化机理,需要藻体内被13 C标记的每类脂质含量的最大化,针对DGDG、PG、SQDG,应选取标记了8天的微藻来投喂生物,而针对MGDG,则应选取标记了10天的微藻投喂生物。     

Determination of a metabolite of nifursol in foodstuffs of animal origin by liquid–liquid extraction and liquid chromatography with tandem mass spectrometry

An analytical method has been developed for the detection of a metabolite of nifursol, 3,5‐dinitrosalicylic acid hydrazide, in foodstuffs of animal origin (chicken liver, pork liver, lobster, shrimp, eel, sausage, and honey). The method combines liquid chromatography and tandem mass spectrometry with liquid–liquid extraction. Samples were hydrolyzed with hydrochloric acid and derivatized with 2‐nitrobenzaldehyde at 37°C for 16 h. The solutions of derivatives were adjusted to pH 7.0−7.5, and the metabolite was extracted with ethyl acetate. 3,5‐Dinitrosalicylic acid hydrazide determination was performed in the negative electrospray ionization method. Both isotope‐labeled internal standard and matrix‐matched calibration solutions were used to correct the matrix effects. Limits of quantification were 0.5 μg/kg for all samples. The average recoveries, measured at three concentration levels (0.5, 2.0, and 10 μg/kg) were in the range of 75.8–108.4% with relative standard deviations below 9.8%. The developed method exhibits a high sensitivity and selectivity for the routine determination and confirmation of the presence of a metabolite of nifursol in foodstuffs of animal origin.     

Quantitation of a novel antiemetic (ADR-851) in plasma and urine by reversed-phase high-performance liquid chromatography with fluorescence detection

A sensitive and specific bioanalytical method for quantitation of a novel antiemetic (ADR-851) in plasma and urine has been developed and validated. The drug and internal standard (metoclopramide) are extracted from the plasma matrix by solid-phase extraction on cyanopropyl bonded-phase columns. After extraction, samples are separated by isocratic reversed-phase high-performance liquid chromatography. The parent drug, internal standard and a yet unidentified metabolite are detected by fluorescence. The method requires 1.0 ml of plasma or 0.1 ml of urine and has a lower limit of quantitation of 2 ng/ml with 10.9% relative standard deviation (R.S.D.). Method linearity has been established over a 2-800 ng/ml range when 1.0 ml of plasma is used. The intra- and inter-day imprecisions for the method are typically better than 6% and 11% R.S.D., respectively, in both plasma and urine over the entire dynamic range. The pooled estimate of bias is less than 5% and attests to the excellent accuracy.     

Multiresidue determination of quinolone and fluoroquinolone antibiotics in fish and shrimp by liquid chromatography/tandem mass spectrometry

A multiresidue method was developed to measure low levels of 8 fluoroquinolones (norfloxacin, ofloxacin, danofloxacin, ciprofloxacin, desethylene ciprofloxacin, enrofloxacin, sarafloxacin, and difloxacin) and 4 quinolones (oxolinic acid, flumequine, nalidixic acid, and piromidic acid). Method detection limits range from 0.1 ng/g for quinolones to 0.4 ng/g for fluoroquinolones. Average recoveries range from 57 to 96%, depending on analyte and commodity; relative standard deviations are all less than 18%. The drugs are extracted from tissues using a mixture of ethanol and 1% acetic acid, diluted in aqueous HCI, and defatted by extraction with hexane. The compounds are further isolated using cation-exchange solid-phase extraction and measured using liquid chromatography with electrospray tandem mass spectrometry detection. The method has been evaluated and applied to the analysis of salmon, trout, and shrimp. Detectable residues were observed in 10 out of 73 samples, at concentrations ranging from 0.28 to 16 ng/g.     

Efficient and sensitive screening and confirmation of residues of selected polyether ionophore antibiotics in liver and eggs by liquid chromatography-electrospray tandem mass spectrometry.

A method using liquid chromatography tandem mass spectrometry (LC-MS-MS) with electrospray (ES) for the determination of traces of narasin, monensin and salinomycin in chicken liver and eggs was developed, validated and used for routine surveillance. The essence of this paper is to demonstrate that one single method can serve very well for two entirely different purposes, i.e., screening and confirmation. Highly reliable confirmation of the identity at low concentrations was demonstrated when residues of narasin were detected and quantified (0.2 to 11 ng g(-1)) in 50% of the Swedish eggs analysed in 1999. Four daughter ions were detected with ion ratios meeting suggested confirmation criteria for the European Union, even at 0.2 ng g(-1). The method was found to be highly cost-effective since both screening and confirmation of 98 liver samples were performed in only two analytical runs (the Swedish national surveillance scheme of 1999, report level 5 ng g(-1)). The high performance of the method for the different applications was possible due to a combination of the power of ES-LC-MS-MS, a procedure involving screening of pooled samples, and method optimisation of the work-up (automated solid phase extraction), LC and MS parameters. Validation data for narasin (0.5 to 20 ng g(-1)) in eggs are presented (accuracy 94 to 108%, relative standard deviation 4 to 10%, limit of detection 0.026 ng g(-1)). The time for an LC-MS-MS run was 4 min, corresponding to 48 s per sample in a pool.     

A sensitive and specific method for the determination of total ribavirin in human red blood cells by liquid chromatography-tandem mass spectrometry

A sensitive and specific method using high-performance liquid chromatography (LC)-tandem mass spectrometry (MS) for the analysis of total ribavirin in human red blood cells (RBC) is developed and validated. The method involves the addition of an internal standard and perchloric acid, the conversion of ribavirin phosphorylated metabolites to ribavirin, purification with a solid-phase exchange cartridge, and LC-MS-MS analysis. The MS-MS is selected to monitor m/z 245-113 for ribavirin and m/z 250-113 for [13C]ribavirin using positive electrospray ionization. The calibration curve is linear over a concentration of 100-10,000 ng/mL with a limit of quantitation of 100 ng/mL. Mean interassay accuracy for quality control (QC) at 100, 1000, and 10,000 ng/mL are 101.8%, 99.4%, and 98.8%, respectively. Mean interassay precision (%CV) for QC at 100, 1000, and 10,000 ng/mL are 5.0%, 5.0%, and 2.5%, respectively. Extractibility of total ribavirin from RBC is confirmed with RBC obtained from a [(14)C]ribavirin-dosed monkey. The method is used to determine the free and total ribavirin concentration in human RBC obtained from hepatitis C patients treated with ribavirin.     

Stable Isotope Dilution Analysis of Gibberellin Residues in Tomato Paste by Liquid Chromatography-Tandem Mass Spectrometry

An accurate and sensitive method for the simultaneous determination of gibberellic acid(GA₃), gibberellin A₄(GA₄) and gibberellin A₇(GA₇) residues in tomato paste was developed by coupling solid phase extraction to high performance liquid chromatography-tandem mass spectrometry(LC-MS/MS) with electrospray ionization based stable isotope dilution analysis(SIDA). The isotope labeled internal standard can compensate for the losses during the extraction and cleanup steps and for discrimination due to ion suppression. After extraction from methanol, hydrophile lipophilic balance(HLB) solid phase extraction(SPE) column was tested for the capacity of the cleanup of the tomato paste in compared with C₁₈ SPE column which is the common way to the detection of GAs, and the former gained better result. Spiked experiments were performed in the non-contaminated tomato pastes and the recoveries of GA₃, GA₄ and GA₇ were 42.6%―75.0% in external standard method(ESM) and 91.1%―103.8% in internal standard method(ISM) respectively. The validities of this method were investigated and good analytical performance for the three GAs was obtained, including low limits of method detection(2 ng/g for GA₃ and GA₄, 0.3 ng/g for GA₇), excellent linear dynamic ranges(5―500 ng/g for GA₃ and GA₄, 1―100 ng/g for GA₇) and good relative standard deviation ranges(4.8%―9.4% for the intra-day test and 3.5%―11.9% for the inter-day test).     

Determination of gentamicin in swine and calf tissues by high-performance liquid chromatography combined with electrospray ionization mass spectrometry

Gentamicin is a broad-spectrum aminoglycoside antibiotic widely used in veterinary medicine for the treatment of serious infections. The purpose of this study was to develop and validate a method to determine gentamicin residues in edible tissues of swine and calf. Extraction of gentamicin was performed using a liquid extraction with phosphate buffer containing trichloroacetic acid, followed by a solid-phase clean-up procedure on a CBA weak cation-exchange column. Tobramycin was used as the internal standard. After drying of the eluate, the residue was redissolved and further analyzed by reversed-phase liquid chromatography/electrospray ionization tandem mass spectrometry (MS/MS). Chromatographic separation of the internal standard tobramycin and the gentamicin components was achieved on a Nucleosil (5 microm) column using a mixture of 10 mM pentafluoropropionic acid in water and acetonitrile as the mobile phase. The gentamicin components C1a, C2 + C2a and C1 could be identified with the MS/MS detection, and subsequently quantified. The method was validated according to the requirements of the EC at the maximum residue limit (MRL) (100 ng g(-1) for muscle and fat, 200 ng g(-1) for liver and 1000 ng g(-1) for kidney), half the MRL and double the MRL levels. Calibration graphs were prepared for all tissues and good linearity was achieved over the concentration ranges tested (r > 0.99 and goodness of fit <10%). Limits of quantification of 25.0 ng g(-1) were obtained for the determination of gentamicin in muscle, fat, liver and kidney tissues of swine and calf, which correspond in all cases to at least half the MRLs. Limits of detection ranged between 0.5 and 2.5 ng g(-1) for the tissues. The within-day and between-day precisions (RSD) and the results for accuracy fell within the ranges specified. The method was successfully used for the determination of gentamicin in tissue samples of swines and calves medicated with gentamicin by intramuscular injection.     

Determination of 22 triazole compounds including parent fungicides and metabolites in apples, peaches, flour, and water by liquid chromatography/tandem mass spectrometry

A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed for the determination of 14 parent triazole fungicides and 8 of their metabolites found in apples, peaches, flour, raw water, and tap water. The triazole fungicides chosen for this multiresidue method development project included propiconazole, fenbuconazole and its RH-9129 and RH-9130 metabolites, cyproconazole, difenoconazole, tebuconazole and its HWG 2061 metabolite, hexaconazole, bromuconazole (both stereoisomers), epoxiconazole, tetraconazole, triticonazole and its RPA-404886 and RPA-406341 metabolites, triadimefon, triadimenol, and myclobutanil. Of special concern to the U.S. Environmental Protection Agency were the metabolites common to all triazole fungicides: free triazole, 1,2,4-triazole (T), and its 2 conjugates: triazolylalanine (TA) and triazolylacetic acid (TAA). These metabolites were the primary focus of this project. All samples we cleaned up by a combination of C18 solid-phase extraction (SPE), mixed-mode cationic SPE, and mixed-mode anionic SPE columns. A triple-stage quadrupole mass spectrometer, equipped with electrospray ionization in the positive-ion mode, was used to determine the compounds of interest. T, TA, and TAA were quantitated using isotopically labeled internal standards (IS), in which the 1,2,4-triazole ring had been synthesized by using 13C and 15N (IS_T, IS_TA, and IS_TAA). These isotopically labeled internal standards were necessary to correct for matrix effects. The T, TA, and TAA metabolites were quantitated at the 25-50 parts-per-billion (ppb) level in food commodities and at 0.50 ppb in water. Recoveries were 70-101% from apples, 60-121% from peaches, 57-118% from flour, 75-99% from raw water, and 79-99% from tap water.     

Pharmacokinetics and metabolism of penindolone in rat plasma using liquid chromatography–tandem mass spectrometry

Penindolone (PND) is a novel influenza A virus dual inhibitor that blocks hemagglutinin-mediated adsorption and membrane fusion. A sensitive and specific ultra-performance liquid chromatography–tandem mass spectrometry method was developed and validated to determine PND in rat plasma. Plasma sample preparation was a simple deproteinization with acetonitrile followed by centrifugation. Chromatographic separation was performed on a C₁₈ column with a gradient mobile phase of acetonitrile–water containing 0.1% formic acid. Detection was carried out by electrospray ionization in negative ion multiple reaction monitoring mode. Linear detection responses were obtained for PND ranging from 1 to 1,000 ng/ml. The intra- and inter-day precision (relative standard deviations, RSD) were within 6.5%, and accuracy (relative error, RE) was within ±11.0%. The extraction recovery data for PND and internal standard (IS) were >96.0%. PND was proved to be stable during the sample storage, preparation and analytic procedures. The validated method was successfully applied to pharmacokinetic and bioavailability studies for PND in rats. The results showed the existence of twin peaks, gender difference and nonlinear pharmacokinetics for PND. In addition, two oxidation metabolites and three glucuronidation metabolites of PND were detected by ultra-high-performance liquid chromatography–high resolution mass spectrometry.