A Sensitive LC-MS/MS Method for the Determination of Afatinib in Human Plasma and Its Application to a Bioequivalence Study.

A high performance liquid chromatography-tandem mass spectrometry assay for the determination of afatinib (AFT) in human plasma was established. A simple sample preparation of protein precipitation was used and separation was achieved on a C18 column by the gradient mixture of mobile Phase A of water (containing 0.1% ammonia) and the mobile Phase B of acetonitrile and water (V:V = 95:5, containing 0.2% ammonia). The multiple reaction monitoring mode was used to monitor the precursor-to-production transitions of m/z 486.2 â†’ m/z 371.4 for AFT and m/z 492.2 â†’ m/z 371.3 for AFT-d6 (internal standard) at positive ionization mode. The calibration curve ranged from 0.100 to 25.0 ng·mL-1 and the correlation coefficient was greater than 0.99. The intra- and inter-batch precision was less than or equal to 10.0%. Accuracy determined at four concentrations was in the range of 92.3-103.3%. In summary, our method was sensitive, simple and reliable for the quantification of AFT and was successfully applied to a bioequivalence study.

UPLC-MS/MS for the Simultaneous Determination of Sildenafil and N-Desmethyl Sildenafil.

A sensitive high-performance liquid chromatography-tandem mass spectrometry method was established for the simultaneous determination of sildenafil and N-desmethyl sildenafil in human plasma. The protein precipitation was used for extraction and the gradient elution of the mobile phase A of water (containing 0.01% formic acid) and the mobile phase B of acetonitrile, and methanol (V:V = 1:1, containing 0.01% formic acid) was used for chromatographic separation on a C18 column. Quantification was performed by multiple reaction monitoring mode to monitor the precursor-to-product ion transitions of m/z 475.4 â†’ m/z 283.3 for sildenafil, m/z 461.4 â†’ m/z 283.2 for N-desmethyl sildenafil, m/z 483.3 â†’ m/z 108.1 for sildenafil-d8 (IS) and m/z 469.2 â†’ m/z 283.3 for N-desmethyl sildenafil-d8 (IS) at the positive ionization mode. The intra- and inter-day relative standard deviations were less than 6.8% and 4.1% for sildenafil and N-desmethyl sildenafil, respectively. Accuracy at four levels ranged from 93.1% to 115.9% for sildenafil and 95.6% to 112.5% for N-desmethyl sildenafil. The present method was sensitive and reliable for simultaneous quantification of sildenafil and its active metabolite and was successfully applied to a pharmacokinetic study of an oral low dose of sildenafil in Chinese healthy volunteers.

Simultaneous determination and determination of sildenafil and its active metabolite in human plasma using LC-MS/MS method.

A sensitive and selective high-performance liquid chromatography-tandam mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous quantification of sildenafil and its metabolite N-desmethyl sildenafil in human plasma. Sildenafil-d8 was used as an internal standard. The analytes were extracted by precipitation extraction and chromatographed on a C18 column using mobile phase A of water (containing 0.1% formic acid) and mobile phase B of acetonitrile (containing 0.1% formic acid) with gradient elution. Quantification was done using multiple reaction monitoring mode to monitor the precursor-to-product ion transitions of m/z 475.4 → m/z 283.3 for sildenafil, m/z 461.4 → m/z 283.2 for N-desmethyl sildenafil and m/z 483.3 → m/z 108.1 for IS in positive ionization mode. The calibration curve was established over the range of 2.00-1,000 ng/ml and the correlation coefficient was >0.99. The intra-day and inter-day relative standard deviations were <6.5% for sildenafil and 6.3% for N-desmethyl sildenafil respectively. Accuracy determinaed at four concentrations was 86.50-105.67% for sildenafil and 96.83-114.40% for N-desmethyl sildenafil. This method was successfully applied to a pharmacokinetic description of sildenafil and the effect of food intake on the pharmacokinetics of sildenafil was also demonstrated in healthy Chinese volunteers.

[Correlation of endogenous androgen and androgen receptor level with coronary artery diseases in elderly males].

OBJECTIVE: To investigate the correlation of endogenous androgen and androgen receptor (AR) level with coronary artery diseases (CAD) in elderly males and elucidate the potential mechanism of gender difference in the prevalence of CAD. METHODS: A total of 296 male patients from different centers were divided into the CAD group (n = 237) and the control group (n = 59) according to the results of coronary angiography. Their mean ages were 68.6 ± 6.8 and 66.2 ± 6.5 years old respectively. The serum levels of FT (free testosterone), TT (total testosterone), E2 (estradiol), LH (luteinizing hormone), FSH (follicle-stimulating hormone), SHBG (sex hormone-binding globulin) and DHEA (dehydroepiandrosterone) were measured in all participants. And the androgen receptors of peripheral lymphocytes were assessed by flow cytometry. RESULTS: The serum level of FT was lower in the CAD group than that in the control group [(24.1 ± 22.2) × 10(-9) mmol/L vs (34.1 ± 31.8) × 10(-9) mmol/L, P = 0.06]. But two groups showed no statistic differences in the levels of TT, E(2), LH, FSH, SHBG, DHEA and lymphocyte AR (56.3% ± 24.00 vs 57.1% ± 20.8%). As demonstrated by the logistic regression analysis, the level of FT was negatively correlated with the CAD risk (OR = 0.98, P = 0.0049) and positively correlated with the peripheral lymphocyte AR level. However age was negatively correlated with the levels of FT and AR. CONCLUSION: The deficiency of endogenous androgen contributes to a high prevalence of CAD in elderly males. The age-related decreases of FT and AR impair the physiological functions of androgen so as to accelerate the progression of coronary atherosclerosis.

Effect of aging on the expression of peroxisome proliferator-activated receptor gamma and the possible relation to insulin resistance.

BACKGROUND: The morbidity of insulin resistance tends to increase with aging. However, studies on molecular mechanisms underlying insulin resistance in aging process is still in paucity. OBJECTIVE: The effect of aging on peroxisome proliferator-activated receptor gamma (PPARgamma) expression, and in addition, the possible association of PPARgamma expression with insulin resistance in aging process were investigated. METHODS: The minimal model technique (MMT) based on frequently sampled intravenous glucose tolerance test was adopted and SI and glucose effectiveness of young and aged rats were compared. RT-PCR and Western blot were used to determine the expression of PPARgamma at mRNA and protein level in adipose tissue and skeletal muscle, as well as in human colic omentum, respectively. RESULTS: MMT result implied existence of various degrees of insulin resistance in aged rats. The expression of PPARgamma at both mRNA and protein levels in adipose tissue of aged rats dramatically decreased; consequently, the expression of its target gene lipoprotein lipases mRNA also markedly decreased compared with those in young rats. Furthermore, the level of PPARgamma mRNA and glucose transporter-4 mRNA in skeletal muscle of aged rats attenuated significantly. The expression of PPARgamma mRNA in omental adipose tissue of old men was significantly decreased, accompanied by a tendency of higher insulin resistance index, compared with those of the young. CONCLUSION: Aging may be associated with diminished PPARgamma expression affecting insulin resistance in the aged individuals.

Age-related decrease in expression of peroxisome proliferator-activated receptor alpha and its effects on development of dyslipidemia.

BACKGROUND: Ageing is associated with increased incidence of dyslipidemia. To investigate potential molecular mechanisms, the effects of age and fibrate administration on peroxisome proliferator-activated receptor alpha (PPARalpha) expression in livers of young and old rats were studied. METHODS: A total of 16 young (2-month-old) and 16 old rats (24-month-old) were randomly assigned to a control group and fenofibrate group (fenofibrate in a total therapeutic dosage of 0.5% in ratio to each treated rat weight in 14 days). RT-PCR was applied to evaluate hepatic mRNA expression of PPARalpha and its target genes. Western blotting was used to determine PPARalpha protein level in liver tissue. RESULTS: When compared with 2-month-old rats, the liver tissue from 24-month-old rats showed reduced expression of PPARalpha mRNA (52%, P < 0.05) and protein (109%, P < 0.01). Consequently, the mRNA levels of PPAR target genes, LPL, ACO, ACS and CPT-1 were markedly lowered by 19%, 8%, 13% and 9% respectively, and apoCIII increased by 24% in livers from 24-month-old rats, compared with values obtained from 2-month-old rats (P < 0.05). Fenofibrate therapy significantly lowered plasma triglyceride and total cholesterol levels in old rats, accompanied with improvement in hepatic expression of genes, including LPL, ACO, ACS, CPT-1 and apoCIII, but no change was found in PPARalpha expression in livers from either 24-month or 2-month-old rats. CONCLUSIONS: The decrease in the hepatic PPARalpha expression is probably directly related to the lipid metabolic disturbances observed in old animals. The beneficial effects of fenofibrate administration in old rats suggests that fibrates may be useful for treating lipid disturbances in old people.