Intersex effect of lamotrigine on the pharmacokinetic parameters of CDRI-97/78, a novel trioxane antimalarial compound, in rats

Reports regarding drug toxicity and adverse events resulting from coadministration of multiple drugs are increasing at an alarming rate. CDRI-97/78 is an 1,2,4-trioxane antimalarial agent under development which gets metabolized to the in vivo active metabolite 97/63. In order to assess its drug interaction potential, CDRI-97/78 was administered alone and in combination with lamotrigine to male and female rats via the oral route. Quantification of the active metabolite 97/63 in rat plasma was achieved with an LC-MS/MS assay. After oral administration of 97/78, the Tmax and Cmax values of 97/63 in male rats were 1.75±0.77 h and 862±306 ng/mL while female rats showed values for Cmax of 622.75±95.09 ng/mL and for Tmax of 7.5±0.5 h. Coadministration of 97/78 and lamotrigine resulted in decreased Tmax and Cmax values in both male and female rats (Tmax and Cmax of 0.77±0.16 h and 58.58±6.43 ng/mL in male rats; 1.13±0.22 h and 62.95±12.00 ng/mL in female rats, respectively). A statistically significant difference (P<0.05) was observed for the pharmacokinetic parameters of 97/63 after oral administration of 97/78 alone and upon its coadministration with lamotrigine except for the Cmax and Tmax values in male and for the T1/2 value in female rats. Statistically, no significant difference for the pharmacokinetic parameters of 97/63 between male and female rats after oral administration of 97/78 alone or in combination with lamotrigine was determined except for Tmax. The study indicates that coadministration of 97/78, an antimalarial agent, and the antiepileptic lamotrigine may require dose adjustments. Additional clinical drug interaction trials may be required to confirm these findings.     

Liquid chromatographic tandem mass spectrometric assay for simultaneous quantification of compound 97/78 and its in vivo metabolite 97/63, a novel trioxane antimalarial, in human plasma and its application to a protein binding study

A sensitive, selective and specific LC-MS/ MS assay for simultaneous quantification of compound 97/78 and its active in vivo metabolite 97/63, a novel 1,2,4-trioxane antimalarial, in human plasma has been developed and validated using alpha-arteether as internal standard (IS). Extraction from plasma involves a simple protein precipitation method. The analytes were chromatographed on a Columbus C18 column with guard by isocratic elution with acetonitrile:ammonium acetate buffer (10 mM, pH 4.0) (80:20 v/v) as mobile phase at a flow rate of 0.45 mL min(-1) and analyzed in multiple reaction-monitoring (MRM) positive ion mode. The chromatographic run time was 4.0 min. The weighted (1/x2) calibration curves were linear over a range of 1.56-200 ng mL(-1) with correlation coefficients > 0.998. For both analytes, the limit of detection (LOD) and lower limit of quantification (LLOQ) were 0.5 ng mL(-1) and 1.56 ng mL(-1), respectively. The recovery of 97/78, 97/63 and IS from spiked control samples were > 90% and their matrix suppression obtained were < 8 %. The accuracy (% bias) and precision (%RSD) for both analytes were < 6.78%. Both analytes were stable after three freeze-thaw cycles (% deviation < 12.80), long-term for 30 days in plasma at -60 degrees C (% deviation < 14.38), for 8 h on bench top in plasma at ambient temperature (% deviation < 1.52) and also in the auto-sampler for 12 h (% deviation < 3.9%). The validated method was successfully applied to a protein binding study of compound 97/78 and metabolite 97/63 in human plasma. Furthermore, the validated method will be applicable to pharmacokinetics, bioavailability and metabolism in various clinical phases and in drug interaction studies.     

LC-MS/MS法定量研究比格犬注射liguzinediol的血浆药代动力学

<正>liguzinediol是一种毒性较小[1],具有心脏安全性,可用于治疗急性心衰的化合物。它通过影响肌浆网钙释放,增加大鼠在体和离体的心脏收缩力[2-3],对正常大鼠及其离体心脏起到较好的正性肌力作用[4-5]。前期对liguzinediol在大鼠体内的药代动力学、♀♂差异及药物代谢进行了研究报道[6-8]。根据一类新药研制的要求,比格犬体内的药代动力学研究对指导临床具有更为重要     

LC-MS/MS法研究阿德福韦酯胶囊的人体药动学

目的建立LC-MS/MS法测定血浆中阿德福韦酯活性代谢物阿德福韦的浓度,并用于健康受试者口服阿德福韦酯(E晶型)胶囊后的药动学研究。方法10名受试者口服阿德福韦酯胶囊后,血浆样品经高氯酸沉淀蛋白,LC-MS/MS法测定阿德福韦的浓度,用3P97程序计算主要药动学参数。结果测定血浆中阿德福韦的最低定量限为0.5μg·L-1,血药浓度为0.50~150μg·L-1时质谱响应线性关系良好(r=0.9996)。测得单剂量和稳态时口服10mg阿德福韦酯(E晶型)胶囊后主要药动学参数:ρmax分别为(23.93±6.86)和(38.93±7.52)μg·L-1,tmax分别为(1.5±0.7)和(1.7±0.5)h,t1/2分别为(8.70±1.56)和(10.57±1.16)h,AUC0→48分别为(292.07±62.75)和(486.78±110.65)μg·h·L-1。结论该方法灵敏,可用于阿德福韦酯人体药动学研究。     

LC-MS/MS法测定人血浆中加巴喷丁的浓度及其药动学研究

建立液相色谱串联质谱(LC-MS/MS)法测定人血浆中加巴喷丁的浓度并将其应用于人体药动学研究。取血浆样品经甲醇沉淀蛋白后,以甲醇0.2%甲酸水溶液(80∶20)为流动相,用Inertsil ODS-3 C18柱(50 mm×2.1 mm ID,3μm)分离,采用电喷雾离子源,以多反应监测(MRM)方式进行正离子检测,定量分析的离子反应分别为m/z 172→m/z 154(加巴喷丁)和m/z 130→m/z 71(内标二甲双胍)。加巴喷丁线性范围为40.8～8.16×103 ng.mL 1,定量限为40.8 ng.mL 1,每个样品测试时间仅2.2 min,日内、日间精密度(RSD)均小于12%,准确度(RE)在±6.4%范围内。应用此法研究了20名健康志愿者单剂量口服加巴喷丁胶囊600 mg后的药动学特点。该方法快速、专属、灵敏、适用性强,可应用于加巴喷丁的人体药动学研究。     

HPLC-UV method for assaying 99/357, a synthetic trioxane antimalarial derivative in rat and rabbit serum

In the present study an accurate and precise HPLC-UV assay method in rat and rabbit serum has been developed and validated for determination of 99/357--a new synthetic analogue of artemisinin developed by Central Drug Research Institute (CDRI), of the class of trioxane derivative. Separation was achieved using a C-18 reversed phase column with a mobile phase comprising of acetonitrile and deionized water (80:20%, v/v) using a UV detector, set at a wavelength of 266 nm. The method, applicable to 200 microl serum, involved double extraction of the samples with 20% isopropyl alcohol (IPA) in n-hexane. The recovery of 99/357 in the two matrices was >90%. The method was sensitive with a limit of quantitation of 25 ng/ml in both rat and rabbit matrices. Precision and accuracy were within the acceptable limits, as indicated by relative standard deviation (accuracy) varying from -12.7 to 5.7% and bias (precision) values ranging from 0.6 to 11.8%. Moreover, 99/357 was stable in serum up to 30 days of storage at -60 degrees C and after being subjected to three freeze/thaw cycles. The method will be applied to perform pharmacokinetic studies of 99/357.     

LC-MS/MS法用于他喷他多在大鼠体内的药动学研究

目的建立测定大鼠血浆中他喷他多浓度的LC-MS/MS法,用以研究他喷他多在大鼠体内的药动学行为。方法采用甲醇沉淀蛋白法提取血浆中目标成分。色谱条件:色谱柱为DiamonsilC18柱,流动相为甲醇-5 mmol.L-1醋酸铵-乙酸(体积比58∶42∶0.5),流速为0.5 mL.min-1,柱温为室温。以多反应离子监测(MRM)方式,在正离子模式下进行检测,用于定量分析的主要离子对分别为m/z 222.2→107.1(他喷他多),m/z 307.1→220.1(氟康唑)。结果血浆中内源性物质不干扰他喷他多的测定,他喷他多的线性范围为2.0～200μg.L-1,方法的准确度在96.2%～97.5%内,日内、日间精密度均小于15%。结论作者建立的LC-MS/MS法,简单快速、专属性强,可用于他喷他多在大鼠体内的药动学研究。     

A sensitive LC-MS/MS method for the quantification of fluticasone propionate in human plasma

A sensitive and selective LC-(APCI) MS/MS method capable of quantifying fluticasone propionate (FP) at levels down to 10 pg ml(-1) in human plasma is reported. The method was validated over a linear range from 10 to 1000 pg ml(-1) using a previously published solid-phase extraction procedure with a 13C3-labeled internal standard. The inter and intra batch precision (coefficient of variation) and accuracy (% bias) of the quality controls samples (20, 25, 50, 100, 200, 500 and 1000 pg ml(-1)) were less than 15 and 11%, respectively. The method is robust, rapid (analysis time of 2 min), selective and hence is ideally suited for pharmacokinetic investigations involving inhalation of therapeutic doses of FP.     

LC-MS/MS法同时测定洛匹那韦和利托那韦的血浆浓度

建立液相色谱-串联质谱法 (LC-MS/MS) 测定人血浆中洛匹那韦 (LPV)、利托那韦 (RTV) 的浓度。血浆样品经碱化沉淀蛋白后, 经乙酸乙酯液-液萃取, 以甲醇-0.1%甲酸水溶液 (80∶20) 为流动相, Agilent ZORBAX Eclipse XDB-C₁₈ (150 mm × 4.6 mm ID, 5 μm) 柱分离; 采用电喷雾电离源, 以多反应监测 (MRM) 方式进行正离子检测, 用于定量分析的离子对LPV为629.6→155.2, RTV为721.4→268.2, 内标替米沙坦 (TEL) 为515.2→276.2。测定血浆中LPV线性范围为62.5～10 000 ng·mL⁻¹, 检测限为15 pg·mL⁻¹, RTV的线性范围为12.5～2 000 ng·mL⁻¹, 检测限为8 pg·mL⁻¹, r均大于0.99。日内和日间精密度均小于15%, 提取回收率均大于75%。该法选择性强、灵敏度高、重现性好, 能同时快速、准确测定人血浆LPV和RTV浓度, 为临床治疗药物浓度监测 (TDM) 奠定基础。     

A multi-analyte LC-MS/MS method for screening and quantification of nitrosamines in sartans

An incident of sartan medicine contamination was notified by Europe in June 2018. The contaminant was identified as a probable carcinogenic nitrosamine and the recalls of sartan medicines were soon made. Since then, more nitrosamine contaminants in sartan medicines were reported. To broaden the applicability and variety in nitrosamine determination, a multi-analyte method is required. In this study, a feasible and sensitive multi-analyte LC-MS/MS method for determination of 12 nitrosamines in sartans was established, where the active pharmaceutical ingredients and final products merchandised in Taiwan were also examined. Chromatographic separation was achieved on an Xselect^® HSS T3 column (15 cm × 3 mm i.d., 3.5 μm) with gradient elution using mobile phase A consisting of 0.1% formic acid in water and mobile phase B consisting of 0.1% formic acid in acetonitrile/methanol (2:8). Validation of the proposed method was also carried out. The limit of detection and limit of quantification for 12 nitrosamines were 20 ng/g and 50 ng/g, respectively. The intra-day and inter-day recoveries of nitrosamines were among 80–120% with precision of 20% for most nitrosamines within sartans matrices. The method was successfully established and applied to authentic samples which a total of 98 positive samples containing 5 distinct nitrosamines, including N-nitrosodiethylamine, N-nitrosodimethylamine, N-nitroso-N-methyl-4-aminobutyric acid, N-nitrosomorpholine and N-nitrosopiperidine, were detected from 557 authentic samples.     

A new LC-MS/MS method for the quantification of endogenous and vinyl chloride-induced 7-(2-Oxoethyl)guanine in sprague-dawley rats

Vinyl chloride (VC) is an industrial chemical that is known to be carcinogenic to animals and humans. VC primarily induces hepatic angiosarcomas following high exposures (≥50 ppm). VC is also found in Superfund sites at ppb concentrations as a result of microbial metabolism of trichloroethylene and perchloroethylene. Here, we report a new sensitive LC-MS/MS method to analyze the major DNA adduct formed by VC, 7-(2-oxoethylguanine) (7-OEG). We used this method to analyze tissue DNA from both adult and weanling rats exposed to 1100 ppm [(13)C(2)]-VC for 5 days. After neutral thermal hydrolysis, 7-OEG was derivatized with O-t-butyl hydroxylamine to an oxime adduct, followed by LC-MS/MS analysis. The limit of detection was 1 fmol, and the limit of quantitation was 1.5 fmol on the column. The use of stable isotope VC allowed us to demonstrate for the first time that endogenous 7-OEG was present in tissue DNA. We hypothesized that endogenous 7-OEG was formed from lipid peroxidation and demonstrated the formation of [(13)C(2)]-7-OEG from the reaction of calf thymus DNA with [(13)C(18)]-ethyl linoleate (EtLa) under peroxidizing conditions. The concentrations of endogenous 7-OEG in liver, lung, kidney, spleen, testis, and brain DNA from adult and weanling rats typically ranged from 1.0 to 10.0 adducts per 10(6) guanine. The exogenous 7-OEG in liver DNA from adult rats exposed to 1100 ppm [(13)C(2)]-VC for 5 days was 104.0 ± 23.0 adducts per 10(6) guanine (n = 4), while concentrations in other tissues ranged from 1.0 to 39.0 adducts per 10(6) guanine (n = 4). Although endogenous concentrations of 7-OEG in tissues in weanling rats were similar to those of adult rats, exogenous [(13)C(2)]-7-OEG concentrations were higher in weanlings, averaging 300 adducts per 10(6) guanine in liver. Studies on the persistence of [(13)C(2)]-7-OEG in adult rats sacrificed 2, 4, and 8 weeks postexposure to [(13)C(2)]-VC demonstrated a half-life of 7-OEG of 4 days in both liver and lung.     

Assessment of human deoxynivalenol exposure using an LC-MS/MS based biomarker method

The Fusarium toxin deoxynivalenol (DON) is one of the most abundant mycotoxins worldwide and poses many adverse health effects to human and animals. Consequently, regulatory limits and a provisional maximum tolerable daily intake (PMTDI) for this important type B-trichothecene were assigned. We conducted a pilot survey to investigate the level of DON exposure in Austrian adults by measurements of DON and its glucuronide conjugates (DON-GlcA's), as biomarkers of exposure, in first morning urine. The average concentration of total DON (free DON+DON-GlcA's) was estimated to be 20.4±2.4 μg L?1 (max. 63 μg L?1). Surprisingly, we found that one third of the volunteers (n=27) exceeded the established PMTDI when consuming regular diet. DON-GlcA's were directly quantified by LC-MS/MS and the results were compared with indirect quantification after enzymatic hydrolysis and confirmed the suitability of the direct method. Moreover, we investigated the in vivo metabolism of DON in humans and were able to determine two closely eluting DON-GlcA's in naturally contaminated urine samples for the first time. In contrast to previous findings we have tentatively identified DON-15-glucuronide as a major DON metabolite in human urine based on the analysis of these samples. About 75% of total glucuronides were derived from this metabolite while DON-3-glucuronide accounted for approximately 25%. The reported new findings clearly demonstrate the great potential of suitable biomarkers to critically assess exposure of humans and animals to DON.     

Development of a bioanalytical method for quantification of a 15-mer oligonucleotide at sub-ng/ml concentrations using LC-MS/MS

LC-MS/MS provides a powerful technique for the selective quantification of therapeutic oligonucleotides; however, the LOQ (typically >1 ng/ml) may be higher than desirable for clinical bioanalysis. A method has been developed to allow quantification of a 15-mer unmodified DNA oligonucleotide in human plasma using SPE and UHPLC with MS/MS detection. The LOQ of this assay was 0.05 nM (～250 pg/ml). This method was then further developed by the inclusion of online SPE to increase loading and apply additional sample cleanup. This allowed for improved assay precision at lower concentrations and increased signal, thus allowing the method to be validated over the range of 10-4000 pM (approximately 50-20,000 pg/ml). The method is accurate, precise and selective and it provides proof-of-concept for sub-ng/ml, high-throughput quantification of oligonucleotides using online SPE coupled to ion-pair, reversed-phase LC-MS/MS.     

应用LC-MS/MS法研究茶多酚在大鼠体内的多组分药动学

目的:建立一种LC-MS/MS法同时测定大鼠血浆中茶多酚的4种儿茶素类抗氧化活性成分,进而研究其多组分药动学。方法:选用Hypersil ODS C18柱,甲醇-水(30∶70)为流动相,等度洗脱;ESI离子源,多反应离子监测,负离子扫描。血浆样品经EtOAc液-液萃取;采用峰面积内标法定量。结果:各待测物和内标不受基质干扰,LLOQ达10或5 ng.mL-1;血浆QC样品批内和批间精密度(RSD)<12%,准确度在97%~106%;萃取回收率>72%,基质效应为88%~106%。大鼠iv茶多酚50和100 mg.kg-1后4种儿茶素类的血浆药动学均符合三室模型,酯型儿茶素类的消除半衰期t1/2γ较长,但非酯型t1/2γ较短;4种活性成分的Vd均大于总体水体积。po茶多酚后药时曲线呈双峰或多峰现象,但胆瘘大鼠无此现象;po生物利用度<15%。结论:本实验建立的LC-MS/MS法高度灵敏、特异且精密,茶多酚自深室的消除较慢,分布广泛;po生物利用度较低,药时曲线双峰现象与EHC有关。酯型儿茶素类与非酯型儿茶素类药动学存在明显差异。     

液相色谱-串联质谱法研究白花前胡甲素脂质体在大鼠体内的药动学

目的制备白花前胡甲素脂质体并进一步研究其在大鼠体内的药动学。方法采用乙醇注入法制备白花前胡甲素脂质体,采用正交设计优化处方。白花前胡甲素脂质体按5、10、20mg·kg~(-1)低、中、高3个剂量经大鼠颈静脉给药后检测一定时间点的血药浓度,对照组为10mg·kg~(-1)的白花前胡甲素溶液。结果制备的脂质体含药量为2g·L~(-1),包封率为93.2%,脂质体高、中、低剂量组及对照组的t_(1/2)分别为(136.58±22.86),(74.12±6.97),(44.93±7.47),(51.26±5.13)min;AUC_(0-∞)分别为(215.93±33.24),(91.75±26.47),(29.22±4.47),(66.90±14.54)min·mg·L~(-1)。结论成功制备了白花前胡甲素脂质体,制得的白花前胡甲素脂质体在大鼠体内半衰期显著延长,生物利用度有明显的提高。     

Challenges of developing a bioanalytical method for a macrolide immunosuppressant compound by LC-MS/MS

The quantification of tacrolimus in human whole blood was developed by LC-MS/MS for a range of 50.0 to 50,000.0 pg/ml. Different challenges were faced during method development due to ion-suppression, lack of sensitivity and low recovery. The optimization of the extraction procedure played a crucial role as tacrolimus had to be isolated from red blood cells, to which it is strongly bound. Another particular challenge arose from the freeze-thaw stability where the extracted samples from fresh blood always showed a lower recovery. Finally, matrix effect was observed in some matrices over time, which resulted in a failed long-term stability in whole blood. In order to resolve the matrix effect issue, the sample procedure had to be improved. The final assay showed good recovery, low matrix effect, linearity, blood stability and good precision and accuracy.     

Toxicokinetics of bisphenol A in rats, monkeys and chimpanzees by the LC-MS/MS method

We examined the toxicokinetics of bisphenol A (BPA) in F344 rats, cynomolgus monkeys and chimpanzees. Serum BPA levels were quantified using the LC-MS/MS method. After oral administration at 10 mg/kg, the maximum concentration in the serum (C(max)) and the area under the serum concentration curve (AUC) of BPA in cynomolgus monkeys and chimpanzees were greater than in rats. After oral administration at 100 mg/kg, AUC during the first 4h (AUC(0-->4h)) in cynomolgus monkeys was greater than in rats. In rats, the serum BPA levels were increased again 6h or later after oral administration at each dose, which suggested the enterohepatic circulation of BPA in rats. After subcutaneous administration at 10 mg/kg, the AUCs were ranked in the following order: cynomolgus monkeys>chimpanzees>rats, and C(max) in cynomolgus monkeys was greater than in rats and chimpanzees. After subcutaneous administration at 100 mg/kg to cynomolgus monkeys and rats, both the C(max) and AUCs in cynomolgus monkeys were greater than in rats. In all species, the oral administration of BPA resulted in much lower C(max) and AUCs than subcutaneous administration at the corresponding doses, indicating the low bioavailability of oral administration. This result suggests that BPA undergoes an extensive first-pass metabolism in these animal species. AUCs of subcutaneous administration and the AUC (0-->4h) of oral administration in the two primates were greater than that in rats. Because the systemic clearance for BPA is assumed to be dependent on the hepatic blood flow-rate, the high AUCs in primates are considered to be due to the lower systemic clearance by a lower hepatic blood flow-rate in primates than in rats. In addition, the toxicokinetics of the metabolites of BPA were examined. After the oral administration of 10 mg/kg BPA, both C(max) and AUCs of BPA metabolites were ranked in the following order: cynomolgus monkeys>chimpanzees>rats, and the terminal elimination half-life (T(1/2)) in rats was greater than that in cynomolgus monkeys and chimpanzees, suggesting the enterohepatic circulation of BPA in rats. From these results, the systemic clearance of BPA in primates is considered to be close to that in humans due to the similarity of the hepatic blood flow-rate. Furthermore, the major elimination route of BPA metabolites in primates is assumed to be renal excretion, as in humans, because the enterohepatic circulation that was observed in rats was not observed. In conclusion, primates are thought to be served as a valuable surrogate model for the toxicokinetics of BPA in humans.     

LC-MS/MS法测定尿二棕榈酰磷脂酰胆碱含量的方法学建立及其作为肾损伤标志物的研究(英文)

尿磷脂已被证实是非常灵敏的肾损伤标志物。然而,关于磷脂的研究大部分为定性或相对定量研究。因此,我们建立了定量测定尿中二棕榈酰磷脂酰胆碱(DPPC)的方法学来准确、特异地测定尿中DPPC含量。采用HILIC Silica(2.1 mm×50 mm,5μm)色谱柱,流动相为5 m M的甲酸铵–乙腈进行梯度洗脱,流速0.3 m L/min。该方法在2–200 ng/m L范围内线性良好,精密度、准确性、回收率、稳定性、稀释效应、基质效应等均符合FDA关于生物样本的检测要求。该方法应用于尿中DPPC的含量测定,并对DPPC作为肾损伤标志物的前景进行评价。