Simultaneous determination and pharmacokinetic study of four phenol compounds in rat plasma by ultra-high performance liquid chromatography with tandem mass spectrometry after oral administration of Echinacea purpurea extract.

A rapid and sensitive assay based on ultra-high performance liquid chromatography with electrospray ionization tandem mass spectrometry was established and validated for the simultaneous determination of cichoric acid, chlorogenic acid, quinic acid, and caffeic acid in rat plasma after oral administration of Echinacea purpurea extract using butylparaben as the internal standard. Samples were pretreated by liquid-liquid extraction with ethyl acetate. The separations for analytes were performed on an ACQUITY UPLC HSS C18 column (1.8 µm 2.1 × 100 mm) using a gradient elution program with acetonitrile/10 mM ammonium acetate (pH 5.6) at a flow rate of 0.3 mL/min. The analytes were detected in multiple reaction monitoring mode with negative electrospray ionization. The lower limit of quantification of each analyte was not higher than 10.85 ng/mL. The relative standard deviation of the intraday and interday precisions was less than 14.69%. The relative errors of accuracies were in the range of -13.80 to 14.91%. The mean recoveries for extraction recovery and matrix effect were higher than 80.79 and 89.98%, respectively. The method validation results demonstrated that the proposed method was sensitive, specific, and reliable, which was successfully applied to the pharmacokinetic study of four components after oral administration of Echinacea purpurea extract.

Simultaneous determination of sarpogrelate and its active metabolite in human plasma by liquid chromatography with tandem mass spectrometry and its application to a pharmacokinetic study.

We established a rapid and simple liquid chromatography with tandem mass spectrometry method for the simultaneous determination of sarpogrelate and its active metabolite, M-1, in human plasma. Sarpogrelate, M-1, and the internal standard, ketanserin, were extracted from a 50 µL aliquot of human plasma by protein precipitation using acetonitrile. Chromatographic separation was performed on a Shim-pack GIS ODS C18 column (100 × 3.0 mm; 3 µm) with an isocratic mobile phase consisting of 10 mM ammonium acetate and acetonitrile (70:30, v/v) at a flow rate of 0.6 mL/min; the total run time was <2.5 min. Mass spectrometric detection was conducted in selected reaction-monitoring mode with positive electrospray ionization at m/z 430.35 → 135.10 for sarpogrelate, m/z 330.30 → 58.10 for M-1, and m/z 395.70 → 188.85 for ketanserin. The linear ranges of concentration for sarpogrelate and M-1 were 1-1000 and 0.5-500 ng/mL, respectively. The coefficient of variation for the assay's precision was ≤9.95%, and the accuracy was 90.6-107%. All analytes were stable under various storage and handling conditions, and no relevant crosstalk and matrix effect was observed. This method was successfully applied to a pharmacokinetic study after oral administration of a 100 mg sarpogrelate tablet to healthy male Korean volunteers.

Determination of 12 potential nephrotoxicity biomarkers in rat serum and urine by liquid chromatography with mass spectrometry and its application to renal failure induced by Semen Strychni.

In previous nephrotoxicity metabonomic studies, several potential biomarkers were found and evaluated. To investigate the relationship between the nephrotoxicity biomarkers and the therapeutic role of Radix Glycyrrhizae extract on Semen Strychni-induced renal failure, 12 typical biomarkers are selected and a simple LC-MS method has been developed and validated. Citric acid, guanidinosuccinic acid, taurine, guanidinoacetic acid, uric acid, creatinine, hippuric acid, xanthurenic acid, kynurenic acid, 3-indoxyl sulfate, indole-3-acetic acid, and phenaceturic acid were separated by a Phenomenex Luna C18 column and a methanol/water (5 mM ammonium acetate) gradient program with a runtime of 20 min. The prepared calibration curves showed good linearity with regression coefficients all above 0.9913. The absolute recoveries of analytes from serum and urine were all more than 70.4%. With the developed method, analytes were successfully determined in serum and urine samples within 52 days. Results showed that guanidinosuccinic acid, guanidinoacetic acid, 3-indoxyl sulfate, and indole-3-acetic acid (only in urine) were more sensitive than the conventional renal function markers in evaluating the therapeutic role of Radix Glycyrrhizae extract on Semen Strychni-induced renal failure. The method could be further used in predicting and monitoring renal failure cause by other reasons in the following researches.

[Experimental study of dicholine succinate pharmacokinetics].

We have conducted for the first time an experimental study of pharmacokinetics of dicholine succinate (DCS) for different ways of its administration in rats The quantitative evaluation of DCS and its metabolites was performed by the radioactive isotope technique. Various parameters of DCS pharmacokinetics were estimated, including the dose dependence of drug content in the blood plasma, total bioavailability, distribution kinetics, and the main ways of DCS excretion.

Effect of food intake on the pharmacokinetics of sarpogrelate and its active metabolite following oral administration to beagle dogs.

1. The objectives of this study were to develop a pharmacokinetic model for sarpogrelate and its metabolite M-1 and to identify the effect of food on sarpogrelate and M-1 pharmacokinetics in beagle dogs. 2. A single 100 mg oral dose of sarpogrelate was administered to fasted and fed beagle dogs and the plasma concentrations of sarpogrelate and M-1 were measured simultaneously by liquid chromatography tandem mass spectrometry. The resultant data were analyzed by modeling approaches using ADAPT5. 3. The plasma concentration time course of sarpogrelate and M-1 were described using a parent-metabolite compartment model with first-order absorption and elimination. The systemic exposure of sarpogrelate and its metabolite after the administration of a single 100 mg oral dose was significantly decreased under the fed condition compared to that under the fasting condition. Modeling approaches have sufficiently explained the food effect of sarpogrelate, i.e. an increased Vc and decreased Ka, in fed dogs. The food effect of sarpogrelate was due to its pH-dependent dissolution. 4. These findings suggest that food intake affects both the rate and extent of absorption of sarpogrelate, and that the pharmacological effect of sarpogrelate can differ significantly according to food intake.

Comparison of pharmacokinetics between sarpogrelate hydrochloride immediate-release formulation and controlled-release formulation.

OBJECTIVE: Sarpogrelate hydrochloride is a selective 5-hydroxytryptamine receptor subtype 2A (5HT(2A)) antagonist that blocks serotonin-induced platelet aggregation. The aim of this study was to compare the pharmacokinetics of sarpogrelate and its metabolite after dosing with a controlledrelease (CR) formulation or an immediaterelease (IR) formulation. METHODS: In this open-label, 2-period, 2-treatment crossover study, 36 healthy male subjects were evenly allocated to two groups in a sequence-randomized manner. In the first period, the first group received 100-mg sarpogrelate IR 3 times at a 6-h interval, and the second group received 300-mg sarpogrelate CR once. After a 7-day washout, the two groups switched their dosing schedule. Serial blood sampling was performed up to 24 hours after the first drug administration during each period. Plasma concentrations of sarpogrelate and its metabolite (M-1) were measured using liquid chromatography-tandem mass spectrometry. Pharmacokinetic parameters were calculated by noncompartmental methods. RESULTS: There were no significant differences between two formulations in the pharmacokinetic properties in the time to reach maximum plasma concentration (C(max)) of sarpogrelate and its metabolite. The CR-to-IR geometric mean ratios, as measured by area under the plasma concentration-time curve (AUC) were 1.31 (90% confidence interval, 1.22 - 1.41) for sarpogrelate and 1.21 (1.14 - 1.29) for M-1. The C(max) was 0.98 (0.85 - 1.12) for sarpogrelate and 1.07 (0.96 - 1.19) for M-1. CONCLUSIONS: After the administration of sarpogrelate hydrochloride CR and IR formulations using the same daily dose, AUCs were slightly higher for the CR formulation than for the IR formulation for both sarpogrelate and its metabolite M-1, but the C(max) values were similar.

Biochemical, molecular, and clinical characteristics of children with short chain acyl-CoA dehydrogenase deficiency detected by newborn screening in California.

BACKGROUND: Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is an autosomal recessive inborn error of mitochondrial fatty acid oxidation with highly variable biochemical, genetic, and clinical characteristics. SCADD has been associated with accumulation of butyryl-CoA byproducts, including butyrylcarnitine (C4), butyrylglycine, ethylmalonic acid (EMA), and methylsuccinic acid (MS) in body fluid and tissues. Differences in genotype frequencies have been shown between patients diagnosed clinically versus those diagnosed by newborn screening. Moreover, while patients diagnosed clinically have a variable clinical presentation including developmental delay, ketotic hypoglycemia, epilepsy and behavioral disorders, studies suggest patients diagnosed by newborn screening are largely asymptomatic. Scant information is published about the biochemical, genetic and clinical outcome of SCADD patients diagnosed by newborn screening. METHODS: We collected California newborn screening, follow-up biochemical levels, and ACADS mutation data from September, 2005 through April, 2010. We retrospectively reviewed available data on SCADD cases diagnosed by newborn screening for clinical outcomes. RESULTS: During the study period, 2,632,058 newborns were screened and 76 confirmed SCADD cases were identified. No correlations between initial C4 value and follow-up biochemical markers (C4, EMA or MS levels) were found in the 76 cases studied. We found significant correlation between urine EMA versus MS, and correlation between follow-up C4 versus urine EMA. Of 22 cases where ACADS gene sequencing was performed: 7 had two or more deleterious mutations; 8 were compound heterozygotes for a deleterious mutation and common variant; 7 were homozygous for the common variant c.625G>A; and 1 was heterozygous for c.625G>A. Significant increases in mean urine EMA and MS levels were noted in patients with two or more deleterious mutations versus mutation heterozygotes or common polymorphism homozygotes. Clinical outcome data was available in 31 patients with follow-up extending from 0.5 to 60 months. None developed epilepsy or behavioral disorders, and three patients had isolated speech delay. Hypoglycemia occurred in two patients, both in the neonatal period. The first patient had concomitant meconium aspiration; the other presented with central apnea, poor feeding, and hypotonia. The latter, a c.625G>A homozygote, has had persistent elevations in both short- and medium-chain acylcarnitines; diagnostic workup in this case is extensive and ongoing. CONCLUSIONS: This study examines the largest series to date of SCADD patients identified by newborn screening. Our results suggest that confirmatory tests may be useful to differentiate patients with common variants from those with deleterious mutations. This study also provides evidence to suggest that, even when associated with deleterious mutations, SCADD diagnosed by newborn screening presents largely as a benign condition.

Synthesis, transport and mechanism of a type I prodrug: L-carnitine ester of prednisolone.

Aerosol glucocorticoid medications have become more and more important in treating BA (bronchial asthma). Although these agents are dosed to directly target airway inflammation, adrenocortical suppression and other systematic effects are still seen. To tackle this problem in a novel way, two L-carnitine ester derivatives of prednisolone (as the model drug), namely, PDC and PDSC, were synthesized to increase the absorption of prednisolone across the human bronchial epithelial BEAS-2B cells by the organic cation/carnitine transporter OCTN2 (SLC22A5) and then to slowly and intracellularly release prednisolone. The transport of prednisolone, PDC and PDSC into the human bronchial epithelial BEAS-2B cells was in the order PDSC > prednisolone > PDC at 37 °C. It was found that PDSC displayed 1.79-fold increase of uptake compared to prednisolone. Transport of PDSC by BEAS-2B was temperature-, time-, and Na(+)-dependent and saturable, with an apparent K(m) value of 329.74 µM, suggesting the involvement of carrier-mediated uptake. An RT-PCR study showed that organic cation/carnitine transporters OCTN1 and OCTN2 are expressed in BEAS-2B cells, but little in HEK293T cells. The order of uptake by HEK293T was prednisolone > PDC > PDSC. In addition, the inhibitory effects of organic cations such as L-carnitine, ergothioneine, TEA(+) and ipratropium on PDSC uptake in BEAS-2B cells were in the order L-carnitine > ipratropium > TEA(+) > ergothioneine, whereas their inhibitory effects on PDSC uptake in HEK293T cells were negligible. Finally, in vitro LPS-induced IL-6 production from BEAS-2B was more and longer suppressed by PDSC than prednisolone and PDC. All of these results suggested PDSC may be an attractive candidate for asthma treatment.

Liquid chromatography tandem mass spectrometry method for the quantification of sarpogrelate, a selective 5-HT(2A) receptor antagonist, in plasma: application to a pre-clinical pharmacokinetic study.

A simple LC-MS/MS method was developed and validated for the estimation of sarpogrelate in 50 µL of rat plasma. The analyte and internal standard (IS) were extracted from rat plasma by acetonitrile precipitation and they were separated on a reversed-phase C8 column with gradient program. The MS acquisition was performed with multiple reaction monitoring mode using m/z 430.2 to m/z 135.0 for analyte and m/z 448.2 to m/z 285.3 for IS. The calibration curves were linear over the range of 1-1000 ng/mL with the correlation coefficient greater than 0.999. With dilution integrity up to 20-fold, the upper limit of quantification was extendable up to 15,000 ng/mL. The method was successfully applied to the analysis of rat plasma samples after single dose oral administration of sarpogrelate at 5 mg/kg to rats for the determination of its pharmacokinetics. Following oral administration the maximum mean concentration in plasma (C(max), 11514 ng/mL) was achieved at 0.25 h (T(max)) and the area under curve (AUC0-24) was 11051 ± 3315 ng h/mL. The half-life (t(¹/2)) and clearance (Cl) were 2.9 ± 1.1 h and 490 ± 171 mL/h/kg, respectively. We believe that development of a method in rodent plasma would facilitate the ease of adaptability of sarpogrelate in human plasma.

Chicoric acid (CA) induces autophagy in gastric cancer through promoting endoplasmic reticulum (ER) stress regulated by AMPK.

Gastric cancer is one of the most common cancers leading to tumor-related deaths worldwide. Chicoric acid (CA) exhibits a variety of protective effects in different diseases. However, its role in regulating tumor progression has not been reported. Autophagy, as a conserved catabolic process, sustains cellular homoeostasis responding to stress to modulate cell fate. In the study, the effects of CA on gastric cancer were investigated. The results indicated that CA treatment markedly reduced the cell viability and induced apoptosis in gastric cancer cells, and prevented tumor growth in an established xenograft gastric cancer model. Furthermore, CA exposure significantly induced autophagy both in gastric cancer cells and tumor samples, as evidenced by the up-regulated expression of LC3II. Moreover, phosphorylated AMP-activated protein kinase (AMPK) and p70S6 kinase (p70s6k) expression were obviously promoted by CA in vitro and in vivo. Importantly, blocking AMPK activation abrogated CA-induced expression of LC3II in gastric cancer cells. In addition, endoplasmic reticulum (ER) stress in tumor samples or cells was markedly induced by CA treatment through promoting the expression of associated signals such as Parkin, protein kinase RNA-like ER kinase (PERK), activating transcription factors 4 (ATF4) and ATF6. Importantly, these effects were abolished by the inhibition of AMPK signaling. Collectively, our findings indicated that CA prevents human gastric cancer progression by inducing autophagy partly through the activation of AMPK, and represents an effective therapeutic strategy against gastric cancer development.

Accumulation of methylguanidine and changes in guanidino compound levels in plasma, urine, and kidneys of furosemide-treated rats.

Antidiuresis and renal diseases alter the levels of guanidino compounds (GCs) in various tissues. Therefore, we hypothesized that diuresis could also disturb GC metabolism, storage, and elimination. In this study, rats were made diuretic to analyze GC levels in plasma, urine, and kidneys. Furosemide was chosen because of its wide use in various human pathologies. Rats were injected intraperitoneally 5 or 10 mg furosemide spread over a 24-hour cycle. Urine was collected over a period of 24 hours before and during furosemide treatment. Plasma was obtained from arterial blood. Renal zones were dissected. The GCs were determined by liquid chromatography. Five milligrams of furosemide provoked a significant increase in plasma and urine levels of GCs compared with those of the controls. The renal distribution and content of GCs were weakly modified by furosemide except for methylguanidine (MG). The level of MG was enhanced by 10 to 16 times in all renal zones. The MG level was 60% higher in renal zones of rats treated with 10 rather than 5 mg furosemide. The fractional excretion of MG was decreased by furosemide. Our data suggest that MG accumulation in kidney and plasma was caused by furosemide, which might induce MG synthesis, and that MG washout from tissue cells into urine by furosemide through the kidney may cause an increase in MG in the kidney.

Guanidino compounds after creatine supplementation in renal failure patients and their relation to inflammatory status.

BACKGROUND: Specific guanidino compounds have been described as uraemic toxins and their concentrations are increased in renal failure due to dimished glomerular filtration, whereas the guanidino compound creatine is used as a performance-enhancing substance in athletes. The present study investigates the effects of creatine supplementation on plasma guanidino compounds in a chronic haemodialysis population. METHODS: Twenty male haemodialysis patients were included in a placebo-controlled cross-over trial. Patients were treated with creatine (2 g/day) or placebo during two treatment periods of 4 weeks, separated by a washout of 4 weeks. Plasma guanidino compounds and routine biochemical parameters were determined, as well as the prognostic inflammatory and nutritional index (PINI). RESULTS: Upon creatine supplementation, guanidinoacetate concentrations decreased by 15%, due to inhibition of creatine synthesis. Concentrations of alpha-keto-delta-guanidinovaleric acid increased three-fold and argininic acid concentrations doubled. Guanidinosuccinate concentrations did not change, but correlated inversely with CRP (r = -0.736; P = 0.001), PINI-score (r = -0.716; P = 0.002) and correlated positively with plasma urea concentration (r = 0.54; P = 0.02). CONCLUSIONS: Creatine supplementation in haemodialysis patients significantly altered the concentration of specific guanidino compounds. Guanidinosuccinate correlated positively with plasma urea and negatively with inflammation markers.

Assay of methylmalonic acid in the serum of patients with cobalamin deficiency using capillary gas chromatography-mass spectrometry.

To determine the incidence of elevated levels of serum methylmalonic acid in patients with cobalamin deficiency, we utilized a new capillary gas chromatographic-mass spectrometric technique to measure methylmalonic acid in the serum of 73 patients with clinically confirmed cobalamin deficiency. Values ranged from 55 to 22,300 ng/ml, and 69 of the 73 patients had values above the normal range of 19-76 ng/ml as determined for 50 normal blood donors. In the cobalamin-deficient patients, serum methylmalonic acid was significantly correlated with the serum folate level and the degree of neurologic involvement. Some patients with pernicious anemia who were intermittently treated with cyanocobalamin were found to have elevated serum levels of methylmalonic acid while free of hematologic and neurologic abnormalities. A cobalamin-deficient patient is described with a normal serum cobalamin and an elevated serum methylmalonic acid. We conclude that the ability to measure methylmalonic acid in human serum will be useful in studies designed to determine the incidence of cobalamin deficiency in various patient populations.

Inhibition of cobalamin-dependent enzymes by cobalamin analogues in rats.

To determine which parts of the cobalamin (cbl) molecule are required for enzyme activity and which parts, if altered, might inhibit cbl-dependent enzyme activity, we synthesized 16 cbl analogues and administered them to nutritionally normal rats. The cbl analogues, with either modifications of the propionamide side chains of the A-, B-, and C-rings, the acetamide side chain of the B-ring, or the nucleotide moiety, were administered to rats by continuous 14-d subcutaneous infusion. Infusion of cbl-stimulated, cbl-dependent activity. Changes in any part of the cbl molecule always abolished stimulation and, in some cases, caused potent inhibition of both cbl-dependent enzymes. The most inhibitory analogues, OH-cbl[c-lactam], a B-ring analogue, and OH-cbl[e-dimethylamide] and OH-cbl[e-methylamide], two C-ring analogues, decreased mean liver holo-L-methylmalonyl-coenzyme A mutase activity to 65% of control values and increased serum methylmalonic acid concentrations to as high as 3,200% of the control values. Liver methionine synthetase activity was decreased to approximately 20% of the control and mean serum total homocysteine concentrations were increased to 340% of control. A similar level of inhibition was demonstrated in rats who were exposed to 28 d of inhaled nitrous oxide or a prolonged period of dietary cbl deficiency. The inhibitory cbl analogues, nitrous oxide, and diet deficiency all depleted liver cbl. The naturally occurring cbl analogues with modifications of the nucleotide moiety had no effects. We conclude that all parts of the cbl molecule are necessary for in vivo cbl-dependent enzyme activity and that modifications of the side chains of the B and C rings are associated with potent in vivo inhibition of cbl-dependent enzyme activity.

Multiple-dose pharmacokinetics and safety of bevirimat, a novel inhibitor of HIV maturation, in healthy volunteers.

BACKGROUND AND OBJECTIVE: Bevirimat [3-O-(3',3'-dimethylsuccinyl)-betulinic acid] is a novel inhibitor of HIV-1 maturation. This study was performed to investigate the pharmacokinetics and safety of bevirimat during repeated dosing in healthy volunteers. SUBJECTS AND METHODS: The study was a 10-day, randomised, double-blind, placebo-controlled, dose escalation study. A total of 48 healthy male volunteers, aged 19-54 years, took part in the study. Treatment was administered for 10 days in six escalating dose cohorts (n = 8 in each cohort; 6 bevirimat, 2 placebo). The doses of bevirimat given in each successive cohort were 25 mg, 50 mg, 75 mg (with 150 mg loading dose), 100 mg, 150 mg and 200mg. Safety follow-up was performed 28 days after the first dose. PHARMACOKINETIC AND STATISTICAL ANALYSIS: Plasma bevirimat levels were measured from blood samples collected pre-dose on days 1-10 and then at approximately 48-hour intervals until 21 days after dosing started. On days 1 and 10, further blood samples were obtained at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8 and 12 hours after dosing. Urine samples were collected in the morning on days 1, 5 and 11 and at the end of the study for the measurement of cortisol and 6beta-hydroxycortisol. The pharmacokinetic parameters of bevirimat were estimated using non-compartmental methods. MAIN OUTCOME MEASURE: Dose proportionality of exposure to bevirimat, assessed by the maximum plasma concentration and the area under the plasma concentration-time curve. RESULTS: The mean terminal elimination half-life of bevirimat ranged from 56.3 to 69.5 hours, and the mean clearance ranged from 173.9 to 185.8 mL/hour. Bevirimat showed approximately 4-fold greater accumulation on day 10 compared with day 1, and the degree of accumulation was similar with all doses. Maximum plasma concentrations ranged from 8 to 58 microg/mL at day 10. Testing for dose-proportionality showed that exposure to bevirimat was proportional to the dose, both after a single dose and after repeat dosing for 10 days. Measurement of the urinary 6beta-hydroxycortisol/cortisol ratio indicated that bevirimat did not affect cytochrome P450 3A activity. Repeated dosing with bevirimat for 10 days was well tolerated. There was no increase in adverse events observed for bevirimat compared with placebo, and no serious adverse events occurred. No clinically relevant changes in vital signs, physical examination or clinical laboratory evaluations were observed. CONCLUSIONS: Bevirimat shows dose-proportional pharmacokinetics during repeated dosing for 10 days. Its accumulation is approximately 4-fold greater on day 10 compared with day 1. Repeated dosing with bevirimat is well tolerated. These properties make bevirimat potentially suitable for inclusion in highly active antiretroviral therapy regimens.

Circulating succinate is elevated in rodent models of hypertension and metabolic disease.

BACKGROUND: Recent evidence suggests that succinate, long known as an intermediate in the citric acid cycle, may also have a role as a signaling molecule through GPR91 and that activation of this receptor results in blood pressure (BP) elevation via the renin-angiotensin system. We sought to test the hypothesis that GPR91 contributes to BP elevation in hypertension. In addition we investigated whether elevated succinate in diabetes could contribute to the increased rate of gluconeogenesis in that condition. METHODS: Circulating succinate concentration was measured using liquid chromatography tandem mass spectrometry in rodent models of hypertension and metabolic disease as well as in human hypertensives and type 2 diabetics in comparison to control subjects. RESULTS: Elevated succinate was detected in spontaneously hypertensive rats (SHR), ob/ob mice, db/db mice, and fa/fa rats in comparison to their non-diseased controls. The changes in concentration are consistent with activation of GPR91. In contrast, neither human hypertensives nor diabetic patients had elevated succinate in comparison to controls. CONCLUSIONS: These findings are consistent with a role of GPR91 signaling in rodent hypertension and diabetes models but not in the analogous human diseases.

Species-dependent plasma metabolism of the ester compound daidzein 7,4'di-succinic acid mon-ester-O-ethoxy (DZ5).

Daidzein 7,4'di-succinic acid mon-ester-O-ethoxy (DZ5) is an ester-containing compound, which was recently synthesized. The objective of this study was to determine the hydrolysis rate of DZ5 in blood from the rat and the dog. The data showed that the hydrolysis rate of DZ5 in plasma was much more rapid in rats than in dogs following intravenous administration. Moreover, similar results were observed after in vitro incubation of DZ5 in the rat or dog plasma. The findings suggested that plasma esterases in the rat plasma have higher activity than in the dog and plasma metabolism of DZ5 is species-dependent.

Validation of an HPLC method for the simultaneous determination of DZ5 and its active metabolite in dog plasma and its pharmacokinetics.

A validated HPLC method is described for the simultaneous determination of daidzein 7,4'-di-succinic acid mon-ester-O-ethoxy (DZ5) and its active metabolite daidzein 7,4'-dioxy-ethoxy (DZ4) in dog plasma. DZ5 and DZ4 were determined by reversed-phase HPLC (column: Hypersil C18 5 microm silica, 200 mm x 4.6 mm i.d.; eluent: 400 ml water, 500 microl 85% phosphoric acid, 600 ml methanol) and photometric detection (250 nm), with Kaempferol as the internal standard. The calibration curve was linear over the range 0.1-50.0 microg/ml in dog plasma. The average extraction recoveries were 84.6% (DZ5), 82.7% (DZ4) and the within-day and between-day precisions were less than 10.93%. The assay was applied to the analysis of samples from a pharmacokinetic study. Following the oral administration and intravenous administration of DZ5, DZ5 was eliminated rapidly from the plasma and DZ4 was found in plasma. The absolute bioavailability of total DZ5 and DZ4 was 41.5%. The method was demonstrated to be feasible for pharmacokinetic studies of DZ5 in dogs.

Simultaneous determination of curcumin diethyl disuccinate and its active metabolite curcumin in rat plasma by LC-MS/MS: Application of esterase inhibitors in the stabilization of an ester-containing prodrug.

Four esterase inhibitors, ethylenediamine tetraacetic acid disodium (Na2EDTA), sodium fluoride (NaF), bis(4-nitrophenyl) phosphate (BNPP) and phenylmethanesulfonyl fluoride (PMSF), were evaluated for their inhibitory effects on enzymatic hydrolysis of labile phenolate esters in curcumin diethyl disuccinate (CDD), a prodrug of curcumin (CUR), in rat plasma. BNPP and PMSF at 10mM exhibited stabilization by preventing degradation of CDD. BNPP at a final concentration of 10mM was subsequently selected to prevent ex vivo metabolism of CDD throughout LC-MS/MS analysis of CDD and CUR in rat plasma. A simple protein precipitation technique using acetonitrile as a precipitating agent was used to extract CDD, CUR and dimethylcurcumin (DMC), an internal standard, from rat plasma. Chromatographic separation was performed on a Halo C8 column (4.6×50mm, 2.7µm) using an isocratic mobile phase containing acetonitrile-0.2% formic acid in water (73:27v/v) with a flow rate of 0.4mLmin(-1). An AB SCIEX QTRAP(®) 6500 mass spectrometer was operated using a positive ion electrospray mode for ionization and detection of analytes and internal standard. Calibration curves for CDD and CUR were established using 50µL of rat plasma over the concentration range of 1-500ngmL(-1). The developed method was fully validated according to US Food and Drug Administration (FDA) guidelines for selectivity, sensitivity, linearity, accuracy, precision, dilution integrity, recovery, matrix effect, and stability. The validated method was applied to evaluate the pharmacokinetics of CDD and CUR in rats after a single intravenous dose of 40mgkg(-1). The method using BNPP as an esterase inhibitor was successful in determining the remaining CDD in rat plasma. The pharmacokinetic results indicate that CDD in rats is converted instantaneously to CUR after intravenous administration and a higher CUR plasma concentration at 5min is achieved in comparison with direct intravenous injection of CUR.

A Novel Simultaneous Determination of Sarpogrelate and its Active Metabolite (M-1) in Human Plasma, Using Liquid Chromatography-Tandem Mass Spectrometry: Clinical Application.

BACKGROUND: This study describes a novel analytical method for simultaneously determining sarpogrelate and its metabolite (M-1) in human plasma, using liquid chromatography coupled with tandem mass spectrometry, with electrospray ionization in the positive ion mode. METHODS: Sarpogrelate, M-1, and labeled internal standard (d3-sarpogrelate) were extracted from 50 µL of human plasma by simple protein precipitation. Chromatographic separation was performed by using a linear gradient elution of a mobile phase involving water-formic acid (99.9:0.1, v/v) and acetonitrile-formic acid (99.9:0.1, v/v) over 4 min of run time on a column, with a core-shell-type stationary phase (Kinetex C18, 50 mm×2.1 mm i.d., 2.6-µm particle size, Phenomenex, USA). Detection of the column effluent was performed by using a triple-quadruple mass spectrometer in the multiple-reaction monitoring mode. RESULTS: The developed method was validated in human plasma, with lower limits of quantification of 10 ng/mL for sarpogrelate and 2 ng/mL for M-1. The calibration curves of sarpogrelate and M-1 were linear over the concentration ranges of 10-2,000 and 2-400 ng/mL, respectively (R(2)>0.99). The carry-over effect, precision, accuracy, and stability of the method met the criteria for acceptance. CONCLUSIONS: A simple, fast, robust, and reliable analytical method was successfully developed and applied to the high-throughput determination of sarpogrelate and its metabolite in real plasma samples in a pharmacokinetic study of healthy subjects.