Metabolism of methoxychlor by hepatic P-450 monooxygenases in rat and human. 1. Characterization of a novel catechol metabolite

Previous investigations demonstrated that the incubation of the chlorinated hydrocarbon pesticide methoxychlor [1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane] with rat liver microsomes generates phenolic estrogenic metabolites. The current study shows that the incubation of liver microsomes from untreated and phenobarbital-treated rats and human donors, in the presence of NADPH, yields three phenolic metabolites. Identification of the metabolites was achieved by TLC, HPLC, GC/MS, and LC/MS and by hydrodynamic voltammetric analysis. These metabolites were identified as the mon- and didemethylated phenolic derivatives (mono-OH-M and bis-OH-M, respectively) and as a novel trihydroxy derivative (tris-OH-M). The tris-OH-M was demonstrated to be a catechol [1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(3,4-dihydroxyphenyl)ethane]. Furthermore, the tris-OH-M becomes radiolabeled by [methyl-3H3]-S- adenosylmethionine (SAM) in a reaction catalyzed by catechol O-methyltransferase (COMT), indicating that tris-OH-M behaves like a catechol. Incubation of the monohydroxy metabolite with liver microsomes from phenobarbital-treated rats (PB microsomes) yields the dihydroxy and the trihydroxy metabolites. Furthermore, the time course of methoxychlor metabolism by PB microsomes demonstrated a rapid appearance and disappearance of the monohydroxy metabolite with the subsequent formation of the dihydroxy and trihydroxy metabolites. On the basis of these findings, it is proposed that the metabolic route of methoxychlor by mono-oxygenases involves sequential demethylations to the dihydroxy derivative and a subsequent ring hydroxylation.     

Characterization of a new rat urinary metabolite of piperine by LC/NMR/MS studies

Potential of piperine, an active alkaloid of black and long peppers, to increase the bioavailability of drugs in humans is of great clinical significance owing to its omnipresence in food. In an attempt to further study the reported differences in its metabolism in rats and humans, a new major urinary metabolite was detected in rat urine and plasma using HPLC. The metabolite was partially purified using reverse phase column chromatography on Sephadex^®-LH 20 and characterized as 5-(3, 4-methylenedioxy phenyl)-2E,4E-pentadienoic acid-N-(3-yl propionic acid)-amide with the help of LC/NMR/positive ESI–MS studies. Complete mass fragmentation pattern could be assigned with MS/MS studies. The metabolite has a unique structure compared to the previously reported metabolites in that it retains methylenedioxy ring and conjugated double bonds while the piperidine ring is modified to form propionic acid group. Mechanism of formation of the metabolite by oxidation and cleavage of piperidine ring is proposed. Kidney appears to be the major excretion route for piperine metabolites in rats as no metabolite could be detected in feces.     

Formation of a toxic metabolite from gentamicin by a hepatic cytosolic fraction.

We have demonstrated recently that incubation of the aminoglycoside gentamicin with an hepatic post-mitochondrial fraction produces a compound toxic to sensory cells from the inner ear in short-term culture; in contrast, the parent aminoglycoside was non-toxic in vitro (Huang MY and Schacht J, Biochem Pharmacol 40: R11-R14, 1990). In the present study, we investigated the subcellular distribution of the enzymatic activity and the nature of the metabolite. Isolated outer hair cells from the guinea pig cochlea were used to assay for cytotoxicity. The enzyme(s) responsible for this novel reaction of aminoglycosides was exclusively localized to the cytosolic fraction of guinea pig liver. No activity was detected in nuclear, lysosomal/mitochondrial or microsomal preparations. Furthermore, the toxin-forming enzymatic activity was associated with the high molecular weight fraction of the cytosol and did not require low molecular weight components. Filtration of the toxin through molecular weight cut-off membranes showed a molecular size of approximately 500. This evidence is consistent with the toxin being a gentamicin derivative.     

Metabolism of the alpha-1A-adrenergic receptor antagonist, pyridine-phenylpiperazine analog (RWJ-69597), in rat, dog and human hepatic S9 fractions -API-MS/MS identification of metabolites.

The In vitro metabolism of the alpha-1A-adrenergic antagonist, RWJ-69597, an analog of pyridine-phenylpiperazines, was conducted after incubation with rat, dog and human hepatic S9 fractions in the presence of an NADPH-generating system. Unchanged RWJ-69597 (> or =43% of the sample in all species) plus 9 metabolites were profiled, quantified, and tentatively identified on the basis of API-MS and MS/MS data. The four metabolic pathways for the formation of RWJ-69597 metabolites are: 1. methyl/phenyl/piperazinylhydroxylation, 2. N/Odealkylation, 3. N-dephenylation, and 4. dehydration. Pathway 1 formed 1 major (8-36%) and 3 minor (<1-3%) hydroxylated metabolites. Pathway 2 produced 2 moderate/minor N/O-dealkylated metabolites (<1- < or =11%), and in conjunction with pathway 1, formed 1 minor diol metabolites (< or =2%). Pathways 3 and 4 generated 2 minor metabolites, N-desphenyl RWJ-69597 (< or =4%) and dehydrated RWJ-69597 (< or =2%), respectively. RWJ-69597 is more extensively metabolized in the rat than the dog or the human in this hepatic system.     

Detection of nimesulide metabolites in rat plasma and hepatic subcellular fractions by HPLC-UV/DAD and LC-MS/MS studies.

Nimesulide (4-nitro-2-phenoxymethanesulfonanilide) is an atypical NSAID lacking a carboxylic acid moiety. It has a good gastric tolerability due to selective inhibition of COX-2. The study objectives in the present work were to characterize the metabolism of nimesulide in rat plasma at certain time intervals. In vitro studies were also carried out to examine if nitroreduction takes place in vitro using rat hepatic subcellular fractions (microsomal and S9 fraction) besides aromatic hydroxylation. This communication describes detection and characterization of nimesulide metabolites isolated from plasma and hepatic subcellular post-incubates by the use of HPLC-UV/diode array and LC-MS/MS. Hydroxynimesulide was the major metabolite both in vivo and in vitro whereas nitroreduction was observed only in vitro with subcellular fractions under anaerobic conditions.     

Quantitative determination of a novel insulin sensitizer and its para-hydroxylated metabolite in human plasma by LC-MS/MS

I, 5-[3-[3-(4-phenoxy-2-propylphenoxy)-propoxy]-phenyl]-2,4-thiazolidinedione sodium salt, is a dual alpha/gamma peroxisome proliferator-activated receptor (PPAR) agonist for potential use in diabetic patients. The compound has a para-hydroxylated metabolite, II, which has also been shown to exhibit PPAR activity. An LC-MS/MS method for the simultaneous determination of I and its active metabolite (II) in human plasma has been successfully developed. The method consists of treating 0.5 ml plasma with ammonium acetate (pH 9.6; 50mM) and extracting I, II and internal standard (III, Fig. 2) with 5 ml ethyl acetate. The ethyl acetate is evaporated and the samples are reconstituted in 0.1 ml acetonitrile:0.1% formic acid (65:35, v/v). The entire extraction procedure, as well as sample collection, was performed in glass tubes and vials to overcome the analytes adherence to polypropylene. A linear HPLC gradient was used to separate the analyte, metabolite, internal standard, and other interfering, non-quantitated metabolites. Detection was by negative ionization MS/MS on a turbo ionspray probe. Precursor-->product ion combinations were monitored in multiple reaction monitoring (MRM) mode. The linear range is 0.05-20 ng/ml for I and 0.1-20 ng/ml for II. Recoveries were 59.4, 90.1 and 56.8% for I, II and III, respectively. Intraday variation using this method was <==7.0% for I and <==9.2% for II. The method exhibits good linearity and reproducibility for each analyte and good sensitivity, selectivity and robustness when used for the analysis of clinical samples.     

Identification of the degradation product of ezlopitant,a non-peptidic substance P antagonist receptor,by hydrogen deuterium exchange,electrospray ionization tandem mass spectrometry (ESI/MS/MS) and nuclear magnetic resonance (NMR) spectroscopy

The degradation product of ezlopitant was isolated from low specific activity material and identified by solution phase hydrogen/deuterium (H/D) exchange and electrospray ionization tandem mass spectrometry (ESI/MS/MS) to be an isopropyl peroxide analog of ezlopitant. The structure of the degradant was further confirmed by nuclear magnetic resonance (NMR) spectroscopy utilizing complete ¹H and ¹³C assignments. Studies were also performed to identify the factors responsible for the oxidative degradation of ezlopitant, which included salt form, storage conditions and salt formation solvent. Of all the variable studies over a 3 weeks period, only a change in the salt form prevented this oxidative degradation.     

Metabolism of a novel CCK-B antagonist in rat,dog and human liver microsomes

1. The in vitro metabolism of a novel CCK-B antagonist ((+)-N-[1-adamantane-1- methyl)-2,4-dioxo-5-phenyl-2,3,4,5-tetrahydro-1H-1,5-benzodiazepin-3-yl]N′-phenylurea; GV150013X) was investigated using rat, dog and human liver microsomes. 2. Four monohydroxy and four dihydroxy metabolites of GV150013X in rat and man were identified by comparison with authentic standards using HPLC and mass spectrometry. 3. The dihydroxy metabolite M1 was not detected in dog liver microsomes mixtures. 4. The formation of dihydroxylated metabolites proceeds via monohydroxylated metabolites M5 and M8 and not directly from GV150013X. 5. A monohydroxy metabolite M5 was the major metabolite in rat and dog, with M5 and dihydroxy metabolites M2 and M3 major metabolites in man.     

Bioactivation of dapsone to a cytotoxic metabolite by human hepatic microsomal enzymes.

1. Using human mononuclear leucocytes as target cells, we have investigated the bioactivation of dapsone (DDS) to a cytotoxic metabolite in the presence of microsomes from nine human livers. Values for NADPH dependent toxicity ranged from 8.8-27% (15.8 +/- 5.9%) and were similar to those for microsomes from control mice, 16-24% (19.0 +/- 4.8%). 2. Microsomes prepared from mice induced with either phenobarbitone or beta-naphthoflavone did not produce significantly more NADPH dependent toxicity than microsomes prepared from control mice. 3. Cytotoxicity was abolished not only by ascorbic acid, but also by sub-physiological concentrations of N-acetylcysteine and glutathione. 4. DDS was metabolised in vitro to a hydroxylamine (metabolic conversion 3.1 +/- 1.5%), which was oxidised further to a cytotoxic metabolite which also became irreversibly bound to protein.     

LC-MS/MS法同时测定大鼠组织中替格瑞洛及代谢物的质量浓度

目的建立LC-MS/MS法同时测定大鼠组织中替格瑞洛及其代谢物AR-C124910XX质量浓度的方法,并应用于替格瑞洛及其代谢物组织分布的研究。方法组织样品采用乙醚提取,Agilent XDB-C_(18)柱分离;流动相为甲醇-乙腈-体积分数0.1%甲酸水溶液(体积比50∶20∶30),流速为0.7 m L·min~(-1);质谱采用MRM多反应检测模式,正离子方式检测。结果替格瑞洛在0.1~50μg·g~(-1)内线性关系良好,日间、日内精密度RSD均不大于12.7%;AR-C124910XX在0.05~20μg·g~(-1)内呈良好线性关系,日间、日内精密度RSD均不大于10.7%。结论该法适合于测定大鼠组织中替格瑞洛及其代谢物的质量浓度,并为替格瑞洛的进一步临床研究奠定基础。     

Characterization of cholate-solubilized dopamine receptors from human, dog and rat brain

[3H]Spiperone binding sites were solubilized in high yield from human, dog and rat brain with a mixture of sodium cholate (0.3% w/v) and sodium chloride (1.4 M). The binding sites were not sedimented after one hour at 100,000 g, they passed freely through 0.20 micron filters, migrated as a single peak in gradient sedimentation and were retarded upon gel filtration, proving that they were truly solubilized. The solubilized binding sites were definitely of dopaminergic nature. They showed saturable, reversible, high affinity binding of [3H]spiperone; displacement of [3H]spiperone binding by nanomolar concentrations of dopamine antagonists and micromolar concentrations of serotonin antagonists; stereo-specificity and a good correlation with drug affinities for membrane preparations. The non-displaceable, non-specific [3H]spiperone binding was very low. Gradient sedimentation analysis revealed a sedimentation coefficient of 12 S for dog solubilized preparations, 9 S for rat solubilized preparations and only 2.5 S for human solubilized preparations (values, uncorrected for detergent binding). Gel filtration experiments seem to confirm these molecular characteristics. Therefore the present results show that the dopamine receptor reveals the same pharmacological properties when solubilized with cholate-salt from rat, dog or human brain, while physico-chemical properties seem to indicate some differences.     

N-glucuronidation of perfluorooctanesulfonamide by human, rat, dog, and monkey liver microsomes and by expressed rat and human UDP-glucuronosyltransferases.

N-Alkylperfluorooctanesulfonamides have been used in a range of industrial and commercial applications. Perfluorooctanesulfonamide (FOSA) is a major metabolite of N-alkylperfluorooctanesulfonamides and has a long half-life in animals and in the environment and is biotransformed to FOSA N-glucuronide. The objective of this study was to identify and characterize the human and experimental animal liver UDP-glucuronosyltransferases (UGTs) that catalyze the N-glucuronidation of FOSA. The results showed that pooled human liver and rat liver microsomes had high N-glucuronidation activities. Expressed rat UGT1.1, UGT2B1, and UGT2B12 in HK293 cells catalyzed the N-glucuronidation of FOSA but at rates that were lower than those observed in rat liver microsomes. Of the 10 expressed human UGTs (1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, 2B15, and 2B17) studied, only hUGT2B4 and hUGT2B7 catalyzed the N-glucuronidation of FOSA. The kinetics of N-glucuronidation of FOSA by rat liver microsomes and by hUGT2B4/7 was consistent with a single-enzyme Michaelis-Menten model, whereas human liver microsomes showed sigmoidal kinetics. These data show that rat liver UGT1.1, UGT2B1, and UGT2B12 catalyze the N-glucuronidation of FOSA, albeit at low rates, and that hUGT2B4 and hUGT2B7 catalyze the N-glucuronidation of FOSA.     

In vitro formation of selegiline-N-oxide as a metabolite of selegiline in human, hamster, mouse, rat, guinea-pig, rabbit and dog.

It is well established in the litrature, that selegiline is metabolised to its N-dealkylated metabolites, N-desmethylselegiline, methamphetamine and amphetamine. However, most studies on selegiline metabolism did not characterize the species differences in the formation of the metabolites. Therefore, in this study, we investigated the in vitro metabolism of selegiline in liver microsomes of different species. In addition, to the previously well-characterized metabolites, selegiline-N-oxide (selegiline-NO) was found to be formed as a metabolite of selegiline in rat liver microsomal preparation. The results of experiments with liver microsomes from other species indicated species differences in the rate and extent of formation of selegiline-NO. The dog and hamster liver microsomal preparations were the most active in terms of selegiline-NO production, whereas little selegiline was metabolized to its N-oxide in human liver microsomes. When selegiline-NO was incubated with rat liver microsomes, no metabolism occurred. When a short incubation time was applied in selegiline expriments no increase in the amount of selegiline-NO was detected. Accordingly, it was clear that selegiline was not metabolized to the N-dealkylated or N,N-bis-dealkylated compounds via selegiline-NO. Studies with different isoenzyme inhibitors indicated that the formation of selegiline-NO might be catalyzed at least partly by cytochrome P450 (CYP) 2D6 and CYP3A4. With the exception of hamster microsomes in the microsomal preparations in vitro, the formation of the R,S-stereoisomer of selegiline-NO was preferred.     

Metabolism of a nitrogen-containing steroid by rat hepatocytes and hepatic subcellular fractions

Freshly isolated rat hepatocytes metabolized the nitrogen-containing steroid N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androst-1-ene-17 beta- carboxamide (I) to six products analyzed by reverse phase HPLC. The major metabolite, which could account for greater than 50% of the total I-related material, exhibited chromatographic, NMR, and mass spectral properties identical to those of the authentic 4-carbinolamide of I. Thus, the major biotransformation pathway in hepatocytes was hydroxylation of the N-methyl group of I to form a stable carbinolamide intermediate of N-demethylation. Desmethyl-I was observed as a minor metabolite. Another metabolite which accounted for approximately 10% of the total I-related material had chromatographic and spectral properties identical to those of the authentic monoethyl analog of I. The other three metabolites were formed in variable quantities and were unstable when isolated. Mass spectral data suggested that one metabolite was the carbinolamide intermediate of N-deethylation. Treatment of rats with phenobarbital or dexamethasone increased the formation of the monoethyl metabolite of I in hepatocytes but had no effect upon the formation of the 4-carbinolamide metabolite. Rat hepatic microsomes catalyzed the NADPH-dependent metabolism of I to the same metabolites in the same relative amounts as observed with intact hepatocytes. Studies with alternative substrates and inhibitors demonstrated that microsomal cytochrome P-450 was responsible for the metabolism of I. Dog and human hepatic microsomes metabolized I to the same products as rat hepatic microsomes, but monoethyl I was the major metabolite.     

Metabolism of a novel antitumor agent, crisnatol, by a human hepatoma cell line, Hep G2, and hepatic microsomes. Characterization of metabolites.

Metabolism of the anticancer agent crisnatol was investigated using a human hepatoma cell line, Hep G2, and human liver microsomes. Crisnatol was metabolized extensively by both systems. The TLC/autoradiographic analysis showed that the crisnatol metabolite profile was similar for both systems and the major metabolites were shown to have structural characteristics similar to those formed by the rat. The Hep G2 cells formed three isomeric dihydrodiols; one of these has been identified by GC/MS and 1H-NMR as the crisnatol 1,2-dihydrodiol. Human liver microsomes also formed two isomeric dihydrodiols with 1,2-dihydrodiol as the major isomer and, in addition, produced 1-hydroxycrisnatol. Crisnatol concentrations of 1.3 micrograms/mL completely inhibited the replication of Hep G2 cells as measured by thymidine incorporation and cell growth kinetics and, at this concentration, cell viability decreased by only 35% as determined by vital staining of cells using neutral red dye.     

Characterization of a novel impurity in bulk drug eprosartan by ESI/MS and NMR

A simple and sensitive liquid chromatography tandem multi-stage mass spectrometry (HPLC/MSⁿ) method suitable for eprosartan analysis was developed, by which an unknown impurity in bulk drug eprosartan was detected. The fragmentation behavior of eprosartan and the impurity in negative mode was investigated. Two molecules of CO₂ lost from eprosartan precursor ion were observed, while four molecules of CO₂ were extruded from the deprotonated molecular ion to the MS³ product ions of the impurity. Furthermore, a characteristic fragmentation ion at m/z 335 was observed in both eprosartan and the impurity indicated that the impurity might have two eprosartan units. The unknown impurity was initially proposed to be eprosartan dimer connected via methylene unit at the thiophene moiety on the basis of the multi-stage mass spectrometric and exact mass evidences, and it was finally elucidated as 4,4′-(5,5′-(1E,1′E)-3,3′-(4,4′-methylenebis(thiophene-4,2-diyl))bis(2-carboxyprop-1-ene-3,1-diyl)bis(2-butyl-1H-imidazole-5,1-diyl))bis(methylene) dibenzoic acid by NMR experiments including 1D (¹H NMR, ¹³C NMR, DEPT135⁰) and 2D (¹H-¹HCOSY, HMQC and HMBC) data.     

HPLC-MS/MS法测定人血浆中阿雷地平及其主要代谢产物的浓度

目的建立高效液相色谱-质谱联用法(HPLC-MS/MS)定量测定人血浆中阿雷地平及其主要代谢产物羟基阿雷地平的浓度。方法人血浆样品用液液萃取法处理,用Kinetex C₁₈柱(4. 6 mm×100 mm,2. 6μm)色谱柱,以纯水-乙腈溶液为流动相,流速为0. 5 mL·min^-1,用电喷雾离子化源,负离子方式,多反应监测(MRM)扫描方式进行监测。考察该方法的专属性、标准曲线与定量下限、精密度与回收率、基质效应和稳定性。结果血浆中阿雷地平的标准曲线方程为y=4. 45×10^-1x+6. 6×10^-3(r=0. 998 2),在0. 02～10μg·L^-1线性关系良好,定量下限为0. 02μg·L^-1;羟基阿雷地平的标准曲线方程为y=9. 82×10^-1x+1. 70×10^-3(r=0. 996 7),在0. 2～100μg·L^-1线性关系良好,定量下限为0. 2μg·L^-1。2种化合物的日内、日间RSD均<10%,提取回收率为91. 78%～97. 97%,无明显的基质效应。结论本法快速、准确、灵敏度高、重现性好,适用于人体血浆中阿雷地平、羟基阿雷地平浓度的测定,可用于阿雷地平、羟基阿雷地平的药代动力学研究。     

Metabolism of 2,6-dinitro[3-3H]toluene by human and rat liver microsomal and cytosolic fractions.

1. 2,6-Dinitrotoluene (2,6-DNT) metabolism by human liver and male Fischer F344 rat liver subcellular fractions under aerobic (100% oxygen) and anaerobic (100% nitrogen) incubation conditions was examined. Under aerobic conditions the major 2,6-DNT metabolite formed by hepatic microsomes was 2,6-dinitrobenzyl alcohol (2,6-DNBalc); under anaerobic conditions 2-amino-6-nitrotoluene (2Am6NT) was the major metabolite. 2. Rates of 2,6-DNBalc formation by human and rat liver microsomes under aerobic conditions were 247 and 132 pmol/min per mg protein, respectively. Rates of 2Am6NT formation by human and rat liver microsomes under anaerobic conditions were 292 and 285 pmol/min per mg protein, respectively. Anaerobic reduction of 2,6-DNT to 2Am6NT by rat and human liver microsomes was inhibited by carbon monoxide and metyrapone, which indicates that microsomal metabolism of 2,6-DNT to 2Am6NT is mediated by cytochrome P-450. 3. Liver cytosolic fractions also metabolized 2,6-DNT to 2Am6NT under anaerobic conditions. Formation of 2Am6NT by human and rat liver cytosols was supported by hypoxanthine, NADPH and NADH. Allopurinol inhibited the hypoxanthine-supported anaerobic metabolism of 2,6-DNT by rat, but not human, liver cytosol. Dicumarol inhibited the NADPH-supported anaerobic metabolism of 2,6-DNT by human, but not rat, liver cytosol. These results indicate that xanthine oxidase contributes to the hypoxanthine-supported anaerobic metabolism of 2,6-DNT by human liver cytosol.     

液相色谱串联质谱法测定人血浆中文拉法辛及其代谢物的浓度

目的建立测定血浆中文拉法辛及其代谢物O-去甲基文拉法辛的液相色谱串联质谱(LC-MS/MS)方法。方法血浆样品中加入内标(氘6-文拉法辛和氘6-O-去甲基文拉法辛),直接沉淀法处理样品。色谱柱为CAPCELL PAK C18 MGⅢ分析柱(100 mm×2.0 mm,5μm),流动相为含0.3%甲酸的水溶液-含0.3%甲酸的乙腈溶液(78∶22,V/V),流速为0.3 mL.min-1。正离子多离子反应监测(MRM)扫描分析,离子通道分别为m/z 278→58(文拉法辛)、m/z 264→58(O-去甲基文拉法辛)、m/z 284→58(氘6-文拉法辛)、m/z 270→58(氘6-O-去甲基文拉法辛)。结果文拉法辛和O-去甲基文拉法辛的线性范围均为2～1 000μg.L-1,定量下限均为2μg.L-1,提取回收率在90.14%～97.33%,批内、批间RSD均小于8%。结论本方法操作简便,特异性强,灵敏度高,可用于人血浆内文拉法辛和O-去甲基文拉法辛的含量测定研究。     

HPLC—MS／MS法测定人体血浆中格列美脲及其活性代谢物

目的：建立同时测定格列美脲（Glimepiride,G）及其活性代谢物羟基格列美脲（Hydroxyl—glimepiride,M1）血浆浓度的高效液相色谱一串联质谱（HPLC—MS／MS）法。方法：血浆样品酸化后,经乙酸乙酯萃取后浓集进样,色谱柱为PhenomenexGeminiC18（50mill×3．0mm,5μm）,甲醇：水：甲酸（80：20：0．1）为流动相,流速为0．2mL／min,柱温为室温,采用电喷雾（ESI）离子源,多反应离子检测模式,以格列齐特（Gliclazide,IS）为内标。G、M1和IS的检测离子对分别为质荷比（仇／z）491．4—352．4、507．4—352．4、324．4—127．2。结果：G和M1的线性范围分别为1．25～400ng／mL和0．313～100ng／mL,最低定量限分别为1．25和0．313ng／mL;批内批间精密度均小于10％,方法回收率均在96．4％～102．5％之间。结论：该方法简便快速、特异性高,可用于同时测定G和M．血药浓度及其人体药代动力学的研究。