Quantification of sedative-hypnotics in human urine and plasma via polystyrene-based solid phase extraction-LC-MS/MS analysis.

Owing to the adverse effects of the overuse of common sedative-hypnotics on human health, the development of an efficient analytical method for the detection of drugs in clinical emergencies and forensic science is significant. Although conventional analytical methods, such as immunoassay, liquid chromatography (LC), gas chromatography, and mass spectrometry (MS) are reliable, they exhibit drawbacks such low-throughput screening and high costs. Thus, in this study, we developed a novel high-throughput method consisting of a polystyrene-based solid phase extraction (SPE) and an LC with tandem MS analysis for the detection of drugs in biological samples and investigated its precision and reliability via the detection of twelve sedative-hypnotics in human urine and plasma samples. Good linear relationship (r ≥ 0.99) were achieved within the concentration range of 0.1-20 ng/mL for the 12 analytes in urine samples. Whereas, in the plasma samples, the correlation coefficient was greater than 0.99 in the concentration range 1-100 ng/mL for lorazepam and clonazepam and in the range 0.5-100 ng/mL for the remaining analytes. The intra- and inter-day precision, autosampler and freeze-thaw stabilities, and lower limit of quantitation (LLOQ) for all twelve analytes in the urine and plasma samples were favorable. Furthermore, sedative-hypnotics were detected in clinical samples obtained from the Hebei General Hospital using this method. These results indicated that the analytical method proposed in this study can be effectively applied in toxicology screening and drug abuse monitoring.The method developed in this study could be applied in clinical and forensic toxicology laboratories for sedative-hypnotic drug screening, providing support for drug abuse monitoring and clinical diagnosis.

Integrating analysis of circular RNA and mRNA expression profiles in doxorubicin induced cardiotoxicity mice.

Doxorubicin (DOX)-induced cardiotoxicity impedes its clinical application, but the mechanisms have not been thoroughly elucidated. Based on circRNA and mRNA expression profiles, we illustrated RNA expression signature changes during DOX-induced cardiotoxicity; mechanism exploration and biomarkers screening were also conducted. Twelve mice were randomly divided into two groups, induction group was treated with doxorubicin, and the control group was given an equal quantity of saline. After the confirmation of myocardial injury in induction group, the heart tissues from both groups were isolated for RNA high-throughput sequencing. The expression profiles were compared between the two groups; a total of 295 mRNAs and 11 circRNAs were shown as biased expression in DOX-induced cardiotoxicity mouse hearts. The dysregulation of three circRNAs were validated by quantitative real-time PCR: mmu_circ_0015773, mmu_circ_0002106, and mmu_circ_001606. In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of the differentially expressed RNAs were performed; the results implied that DOX might cause cardiotoxicity by interfering hemoglobin-based oxygen delivery and DNA-associated signal pathways. We integrated the differential expressed mRNA and validated circRNAs by constructing a competing endogenous RNA (ceRNA) network, which indicated that the alteration of the three circRNAs could activate apoptosis process of myocardial cells. This study provided novel insight into the mechanisms of DOX induced cardiotoxicity, and potential biomarkers or therapeutic targets were also proposed.

Qualitative and quantitative determination of 15 main active constituents in Fructus Sophorae pill by liquid chromatography tandem mass spectrometry.

BACKGROUND: Fructus Sophorae pill, one of the traditional Chinese medicine, was widely used for hemorrhoids, hypertension and odontalgia. This paper describes a sensitive and specific assay for the determination of the 15 active constituents (sophoricoside, genistin, genistein, rutin, quercetin, kaempferol, baicalein, baicalin, naringin, naringenin, hesperidin, neohesperidin, wogonin and cimifugin, prim-O-glucosylcimifugin) in Fructus Sophorae pill. MATERIALS AND METHODS: Chromatographic separation was performed on a C18 column with acidified aqueous methanol gradients at a flow rate of 0.8 mL/min. The identification and quantification of the analytes were achieved by use of a hybrid quadrupole linear ion trap mass spectrometer. Multiple-reaction monitoring scanning was applied to quantification with switching electrospray ion source polarity between positive and negative modes. RESULTS: The proposed method was used to analyze 40 batches of samples with good linearity (r, 0.9990-0.9999), intraday precisions (RSD, 0.14-2.55%), interday precisions (RSD, 0.51-2.81%), stability (RSD, 0.31-2.65%), and recovery (RSD, 1.29-2.95%) of the 15 compounds. In addition, the hierarchical cluster analysis, including a method called furthest neighbor and nearest neighbor, was employed to classify samples according to characteristics of the 15 constituents. CONCLUSION: The results indicated that the analytical method was rapid, reliable, simple and suitable for the quality evaluation of Fructus Sophorae pill.

Pharmacokinetic and excretion study of three secoiridoid glycosides and three flavonoid glycosides in rat by LC-MS/MS after oral administration of the Swertia pseudochinensis extract.

A sensitive, specific and rapid liquid chromatography-mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous determination of swertiamarin, gentiopicroside, sweroside, mangiferin, isoorientin and isovitexin in rat plasma, bile, urine and feces after oral administration of Swertia pseudochinensis extract using sulfamethalazole (SMZ) as an internal standard (IS). The samples were pretreated and extracted by liquid-liquid extraction as the sample clean-up procedure. The chromatographic separation was performed on a C18 column with a linear gradient elution using a mobile phase consisted of 0.1% formic acid and methanol at a flow rate of 0.8 mL/min. The total run time was 10 min. Determination and quantification of the analytes were achieved by use of a hybrid quadrupole linear ion-trap mass spectrometer. A multiple-reaction monitoring scanning (MRM) method with electrospray ionization (ESI) source was employed with simultaneously monitoring the positive and negative electrospray ion source polarity in a single run. A full validation of the method was performed. The linearity of the analytical response was good, with correlation coefficients greater than 0.9953. The intra-day and inter-day precisions (RSD %) exhibited within 10.4%, and the accuracy (RE %) ranged from -8.7% to 9.5%. A non-compartmental model was employed to calculate the parameters. The values of elimination rate constants (Ke) ranged from 0.0026 to 0.0118 and the elimination half-life (T1/2) ranged from 58.4 to 263.0 min. This is the first report on pharmacokinetic and excretion study of Swertia pseudochinensis extract after oral administration. The results provided a meaningful basis for the clinical application of this herb.

Application of a liquid chromatography-tandem mass spectrometry method to the pharmacokinetics, tissue distribution and excretion studies of sweroside in rats.

A sensitive, reliable and accurate high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) was developed and validated for the quantification of sweroside in rat plasma, tissue and excretion. A single-step protein precipitation by methanol was used to prepare samples. Sweroside and swertiamarin (internal standard, IS) were separated by using a C18 column and a mobile phase consisted of methanol and water containing 0.1% formic acid running at a flow rate of 0.8ml/min for 6min. Detection and quantification were performed using a mass spectrometer by the multiple-reaction monitoring (MRM) in positive electrospray ionization mode. The optimized mass transition ion pairs (m/z) for quantitation were [M+H](+)359.1→197.2 for sweroside and [M+Na](+)397.4→165.3 for swertiamarin (IS), respectively. The inter-day precision (RSD %) was less than 11.20% and intra-day precision (RSD %) was less than 10.90%, while the inter-day accuracy (RE %) was ranged from -9.69 to 9.17% and intra-day accuracy (RE %) was ranged from -10.56 to 13.47%. The mean elimination half-life (t1/2) of sweroside for 5, 10 and 15mg/kg dose were 78.8, 67.6 and 77.2min, respectively. And sweroside follows linear plasma pharmacokinetics across the investigated dosage range in rats (5-15mg/kg). The absolute bioavailability (F %) of sweroside was 11.90% on average. The results of tissue distribution showed the higher sweroside concentrations were found in kidney, liver, spleen and lung, and the small amount of drug was distributed into the brain tissue. The high distribution in liver confirms the reports that sweroside has hepatoprotective activity and promoted liver regeneration, and there was no long-term accumulation of sweroside in rat tissues. Total recoveries of sweroside within 48h were 0.67% in bile, 1.55% in urine and 0.46% in feces, which might be resulted from liver first-pass effect. The above results suggested that sweroside was mainly excreted as the metabolites.

Study of in vitro metabolism of m-nisoldipine in human liver microsomes and recombinant cytochrome P450 enzymes by liquid chromatography-mass spectrometry.

This is a report about the investigation of the metabolic fate of m-nisoldipine in human liver microsomes and the recombinant cytochrome P450 enzymes by using LC-MS/MS. A sensitive and reliable LC-MS/MS method was developed to obtain a rapid and complete characterization of new metabolites and the metabolism pathways. The analytes were separated on a reversed phase C18 column with acetonitrile and 0.1% aqueous formic acid as the mobile phase. Tandem mass spectrometry with positive electrospray ionization was used to enable the structural characterization of the metabolites. A total of 10 metabolites were characterized with proposed structures in the incubation of human liver microsomes by comparing their retention times and spectral patterns with those of the parent drug. Dehydrogenation of the dihydropyridine core and reactions of side chains such as hydroxylation and hydrolysis of ester bonds were the major metabolic pathways. The specific cytochrome P450 (CYP) enzymes responsible for m-nisoldipine metabolites were identified using chemical inhibition and cDNA expressed CYP enzymes. The results indicated that CYP2C19 and CYP3A4 might play major roles in the metabolism of m-nisoldipine in human liver microsomes.

Pharmacokinetics and excretion study of sophoricoside and its metabolite in rats by liquid chromatography tandem mass spectrometry.

In this study, a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the determination of sophoricoside and its metabolite genistein in rat plasma, bile, urine and feces after oral administration of sophoricoside, using sulfamethalazole as internal standard (IS). The separation was performed on a reverse phase C18 column with gradient elution consisting of 0.2‰ aqueous formic acid and methanol (containing 0.2‰ formic acid). The detection was accomplished by multiple-reaction monitoring (MRM) scanning after electrospray ionization (ESI) source operating in the negative ionization mode. The optimized mass transition ion pairs (m/z) for quantitation were 431.2/268.2 for sophoricoside, 268.7/133.0 for genistein and 252.0/156.0 for IS. This developed method provides good linearity (r>0.9983), intra- and inter-day precisions (RSD<8.31%) with accuracies (RE, -6.91 to 6.66%), stability (RE, -7.45 to 6.59%), extract recovery (76.24 to 93.30%) and matrix effect (81.06-106.2%) of the analytes in plasma, bile, urine and feces. The mean elimination half-life (t1/2) of sophoricoside and genistein were 59.78±7.19 and 103.14±16.97min, respectively. The results showed that sophoricoside was rapidly absorbed and then eliminated from rat plasma. The total recoveries of sophoricoside in bile, urine and feces were about 0.0111%, 1.76% and 11.13%. The amounts excreted of genistein were 0.42±0.02µg in bile, 10.15±0.22µg in urine and 2.92±0.13µg in feces. This is the first report to evaluate the pharmacokinetics and excretion of sophoricoside and its metabolite in rats after oral administration of sophoricoside monomer. The results provided a meaningful basis for the clinical application of sophoricoside.

Studies on separation and pharmacokinetics of m-nisoldipine enantiomers in beagle dog plasma by LC/MS/MS.

A rapid, sensitive and selective liquid chromatography-tandem mass spectrometric (LC/MS/MS) method was developed and validated for the separation and determination of m-nisoldipine enantiomers in beagle dog plasma. Samples were pretreated by a single-step protein precipitation with acetonitrile. After m-nisoldipine racemic administration to beagle dogs, samples of m-nisoldipine enantiomers in beagle dog plasma were separated and determined on a ULTRON ES-OVM column (150 × 4.6 mm, 5 µm) at 20°C with a mobile phase consisted of methanol-acetonitrile-ammonium acetate (pH 7.0; 2mM) (15:15:70, v/v/v) at a flow rate of 0.8 mL/min. Chromatograms were monitored at 237 nm, and the API 4000 triple quadrupole mass spectrometer was operated in multiple reaction monitoring (MRM) scan mode using ElectroSpray ionization (ESI) source. The good linearity (rs=0.9958 and rr=0.9983) were found in the range 0.25-20 ng/mL. The lower limit of quantification (LLOQ) obtained was 0.25 ng/mL (n=6). All the validation data, such as accuracy, precision, intra-day and inter-day repeatability, were within the required limits. The method was successfully applied to separation and pharmacokinetics of m-nisoldipine enantiomers in beagle dog plasma. The result of statistics analysis shows that there are no significant differences between R-(-)-m-nisoldipine and S-(+)-m-nisoldipine (p>0.05). The study provides necessary evidences for the research and new drug development of m-nisoldipine enantiomers.

Simultaneous determination and pharmacokinetic study of six flavonoids from Fructus Sophorae extract in rat plasma by LC-MS/MS.

In this study, a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the determination of six flavonoids including sophoricoside, genistin, genistein, rutin, quercetin and kaempferol in rat plasma after oral administration of Fructus Sophorae extract using sulfamethalazole as internal standard (IS). The plasma samples were pretreated and extracted by liquid-liquid extraction. Chromatographic separation was accomplished on a C(18) column with a simple linear gradient elution. The detection was accomplished by multiple-reaction monitoring (MRM) scanning after electrospray ionization (ESI) source operating in the negative ionization mode. The optimized mass transition ion pairs (m/z) for quantitation were 431.1/267.9 for sophoricoside and genistin, 269.0/133.0 for genistein, 609.2/300.0 for rutin, 301.0/150.9 for quercetin, 284.9/93.0 for kaempferol and 252.0/155.9 for IS. The total run time was 8.0 min. Full validation of the assay was implemented including specificity, linearity, accuracy, precision, recovery and matrix effect. This is the first report on determination of the major flavones in rat plasma after oral administration of Fructus Sophorae extract. The results provided a meaningful basis for the clinical application of this herb.