Ileal Trichobezoar Presenting with Acute Appendicitis and Intestinal Obstruction: A Rare Case Report and Review of Literature.

Trichobezoar is a rare diagnosis among pediatric patients highlighting underlying psychiatric illness. Gastric bezoar with a long tail extending into small bowel may present with varied presentation including small bowel obstruction. Isolated small bowel trichobezoar is rare making diagnosis difficult highlighted in the index case.

Development of a bioanalytical method for the quantification of sinococuline in human plasma and its application in phase I clinical pharmacokinetic study.

A selective, highly sensitive, precise, and novel bioanalytical method has been developed and validated to quantify sinococuline, an active constituent present in the phytopharmaceutical drug product containing Cocculus hirsutus plant extract, in vivo. Chromatographic separation was achieved on a Luna Omega Polar-C18 bonded analytical column maintained at 45°C. The isocratic mobile phase consisted of methanol and ammonium formate buffer (60:40, v/v) at acidic pH with a low flow rate of 0.250 mL/min. Detection was performed on an API 4000 mass spectrometer using electrospray ionization in positive polarity and multiple reaction monitoring mode to achieve a lower limit of quantification of 1.50 ng/mL. Excellent accuracy and precision were obtained after extracting the analyte from plasma samples using a chemical analogue as an internal standard in the absence of an isotope-labeled compound. The extraction efficacy was evidenced from recovery study, and the analyte was found to be stable in plasma. Validation study demonstrated linearity with coefficient of correlation, r ≥ 0.99, and minimal matrix effect. This bioanalytical method was successfully applied to evaluate pharmacokinetic parameters of sinococuline from a phase I clinical trial of an aqueous extract of C. hirsutus in healthy human volunteers.

Clinical development of imatinib: an anticancer drug.

BACKGROUND: A novel and accurate high-throughput tandem mass spectroscopic method has been developed and validated for determination of imatinib, a protein-tyrosine kinase inhibitor against chronic myeloid leukemia. MATERIALS & METHODS: Chromatographic separation was carried on XTerra® RP18 column (150 mm × 4.6 mm, 5 µm particle size) manufactured by Waters Corporation, MA, USA. The detection was performed on a triple quadruple tandem mass spectrometer by multiple reactions monitoring mode via electrospray ionization source. RESULTS: The selective and sensitive method was linear in the concentration range of 9.57-4513.29 ng/ml and reported no matrix effect. CONCLUSION: The mean Cmax was found to be 10-15% lower in European subjects as compared with Indian subjects.

Histamine H3 receptor antagonists display antischizophrenic activities in rats treated with MK-801.

BACKGROUND: Animal models based on N-methyl-d-aspartate receptor blockade have been extensively used for schizophrenia. Ketamine and MK-801 produce behaviors related to schizophrenia and exacerbated symptoms in patients with schizophrenia, which led to the use of PCP (phencyclidine)- and MK-801 (dizocilpine)-treated animals as models for schizophrenia. METHODS: The study investigated the effect of subchronic dosing (once daily, 7 days) of histamine H3 receptor (H3R) antagonists, ciproxifan (CPX) (3 mg/kg, i.p.), and clobenpropit (CBP) (15 mg/kg, i.p.) on MK-801 (0.2 mg/kg, i.p.)-induced locomotor activity and also measured dopamine and histamine levels in rat's brain homogenates. The study also included clozapine (CLZ) (3.0 mg/kg, i.p.) and chlorpromazine (CPZ) (3.0 mg/kg, i.p.), the atypical and typical antipsychotic, respectively. RESULTS: Atypical and typical antipsychotic was used to serve as clinically relevant reference agents to compare the effects of the H3R antagonists. MK-801 significantly increased horizontal locomotor activity, which was reduced with CPX and CBP. MK-801-induced locomotor hyperactivity attenuated by CPX and CBP was comparable to CLZ and CPZ. MK-801 raised striatal dopamine level, which was reduced in rats pretreated with CPX and CBP. CPZ also significantly lowered striatal dopamine levels, although the decrease was less robust compared to CLZ, CPX, and CBP. MK-801 increased histamine content although to a lesser degree. Subchronic treatment with CPX and CBP exhibited further increased histamine levels in the hypothalamus compared to MK-801 treatment alone. Histamine H3 receptor agonist, R-α methylhistamine (10 mg/kg, i.p.), counteracted the effect of CPX and CBP. CONCLUSIONS: The present study shows the positive effects of CPX and CBP on MK-801-induced schizophrenia-like behaviors in rodents.

Liquid chromatography-tandem mass spectrometry method for the estimation of adefovir in human plasma: Application to a pharmacokinetic study.

An analytical method based on solid phase extraction was developed and validated for analysis of adefovir in human plasma. Adefovir-d4 was used as an internal standard and Synergi MAX RP80A (150 mm×4.6 mm, 4 µm) column provided the desired chromatographic separation of compounds followed by detection with mass spectrometry. The method used simple isocratic chromatographic condition and mass spectrometric detection in the positive ionization mode. The calibration curves were linear over the range of 0.50-42.47 ng/mL with the lower limit of quantitation validated at 0.50 ng/mL. Matrix effect was assessed by post-column infusion experiment to monitor phospholipids and post-extraction addition experiment was performed. The degree of matrix effect for adefovir was determined as 7.5% and ion-enhancement in five different lots of human plasma was 7.1% and had no impact on study samples analysis with 4.5 min run time. The intra- and inter-day precision values were within 7.7% and 7.8%, respectively, for adefovir at the lower limit of quantification level. Validated bioanalytical method was successfully applied to clinical sample analysis.

Metaxalone estimation in biological matrix using high-throughput LC-MS/MS bioanalytical method.

Metaxalone is a skeletal muscle relaxant, an approved drug for pain relief. Published bioanalytical methods lacked detailed stability evaluation in blood and plasma. An accurate, precise, high-throughput tandem mass spectroscopic method has been developed and validated. Following solid phase extraction (SPE), metaxalone and the internal standard metaxalone-d(3) were extracted from an aliquot of 200 µL of human plasma. Chromatographic separation achieved on an Ascentis Express C18 column (50 mm × 4.6 mm i.d., 2.7 µm particle size) with mobile phase is a mixture of 10mM ammonium acetate buffer (pH 4.5)-methanol-acetonitrile (20:50:30, v/v/v), at an isocratic flow rate of 0.7 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The mass transitions of metaxalone and metaxalone-d(3) were m/z 222.3→161.2 and m/z 225.3→163.3, respectively. The linear calibration curves were obtained in the concentration range of 0.105-10.081 µg/mL (r(2)≥0.99) with a lower limit of quantification (LLOQ) of 0.105 µg/mL. The intra- and inter-day precisions and relative error were all within 6%. Despite achieving high mean recovery (>78%), no interference peaks or matrix effects were observed. Detailed stability exercises including drug stability in blood, hemolyzed, lipemic and normal plasma were conducted to extend the method applicability in vast majority of clinical studies using 800 mg metaxalone extended release oral dosage form.

ESI-MS/MS stability-indicating bioanalytical method development and validation for simultaneous estimation of donepezil, 5-desmethyl donepezil and 6-desmethyl donepezil in human plasma.

A bioanalytical method was developed and validated to estimate donepezil, 6-desmethyl donepezil and 5-desmethyl donepezil simultaneously in human plasma using galantamine as an internal standard (IS). The chromatographic separation was achieved on a reverse-phase XTerra RP (150 × 4.6 mm, 5 µm) column without affecting recovery (mean recovery > 60% with CV < 10%) for all analytes. ESI-MS/MS multiple reaction monitoring in positive polarity was used to detect mass pairs for donepezil (m/z 380.3 → 91.3), 6-desmethyl donepezil (m/z 366.4 → 91.3), 5-desmethyl donepezil (m/z 366.4 → 91.3) and galantamine m/z (288.1 → 213.0). The linearity was established over a dynamic range of 0.339-51.870, 0.100-15.380 and 0.103-15.763 ng/mL for donepezil, 6-desmethyl donepezil and 5-desmethyl donepezil, respectively. The current method shows that minimal conversion of labile metabolites to parent donepezil in plasma as stability was successfully achieved for 211 days at -15 °C storage temperature. The method was successfully applied to a clinical study after administration of 10 mg donepezil tablets to healthy male Indian volunteers.

Controlled ex-vivo plasma hydrolysis of valaciclovir to acyclovir demonstration using tandem mass spectrometry.

Plasma estimation of valaciclovir, an antiviral drug, is challenging due to both in-vivo and ex-vivo hydrolysis to active metabolite acyclovir. A simultaneous method is described involving the solid-phase ion-exchange extraction procedure requiring 100 µL of plasma volume, a reverse-phase Lichrosphere RP Select B (125 × 6 mm, 5 µm) column and isocratic mobile phase to achieve the desired chromatographic separation. ESI-MS/MS multiple reaction monitoring in positive polarity, detected mass pairs for valaciclovir (m/z 325.5 → 152.2), acyclovir (m/z 226.3 → 152.2) and respective internal standards valganciclovir (m/z 307.1 → 220.3) and acyclovir-d4 (m/z 230.2 → 152.0). Fully fledged method validation was evaluated as per current regulatory requirements and results were deemed acceptable. The plasma samples showed extensive hydrolysis of valaciclovir when collected or processed at room temperature, without buffer stabilization prior to storage at -15°C. Our results showed that using prechilled K3 EDTA vacutainers immersed in an iced-water bath during blood sample collection, and addition of 50% orthophosphoric acid solution to plasma samples prior to storage at -50°C for at least 120 days controlled the hydrolysis of valaciclovir to acyclovir. While monitoring drug absorption into systematic circulation, the valaciclovir to acyclovir formation ratio was improved to 1:20 in healthy volunteers for the first time.

Bioequivalence of venlafaxine modified-release capsule revisited with an innovative approach using experimental and predictive models.

BACKGROUND: To evaluate the venlafaxine:O-desmethyl venlafaxine (active metabolite) in vivo formation ratio (MR) in three independent bioequivalence (BE) studies consisting of single-dosed (under fasted and fed conditions) and multiple-dosed clinical trials on healthy subjects. The pooled data pharmacokinetic (PK) analysis demonstrates a model to conduct enantiomer/racemate/active metabolite bioanalysis for regulatory submission of bioavailability/bioequivalence (BA/BE) studies using an interesting MR concept. RESULTS: BE was established for all three studies. Moreover, the venlafaxine:O-desmethyl venlafaxine MR for C(max) and AUC(last) differed by more than 50% for fasted and fed single-dosed studies, while pooled data analysis found the MR for C(max) to be approximately 0.63 and the AUC to be approximately 0.36 for both test and reference drugs. However, negligible variation was observed for both rate and extent of drug and active metabolite absorption into the systemic circulation at steady state, as the MR for both C(max) and AUC was approximately 0.62. CONCLUSIONS: The applications/consequences of the above results are immense. First, an achiral assay for venlafaxine and O-desmethyl venlafaxine estimation in human plasma has been justified for the regulatory acceptance of BA/BE studies, supported with both single- and multiple-dosed PK data showing negligible variation in terms of MR at C(max). Second, the current investigation shows the MR to be within ±10% when compared with the single-dosed reported study on a western population. Third, the racial effect would not lead to any significant clinical outcome using an interchangeable venlafaxine 150-mg capsule manufactured by Ranbaxy with an Efexor 150-mg capsule manufactured by Wyeth. Furthermore, a decision tree is proposed to evaluate if a racemate or an enantiomer drug and active metabolite bioanalysis should be executed for BA/BE regulatory submission using respective achiral or chiral assays when the drug moiety is a racemate or an enantiomer, formulated in modified-release dosage forms.

Stability-indicating validation of acitretin and isoacitretin in human plasma by LC-ESI-MS/MS bioanalytical method and its application to pharmacokinetic analysis.

LC- ESI- MS/MS simultaneous bioanalytical method was developed to determine acitretin and its metabolite isoacitretin in human plasma using acitretin-d3 used as the internal standard for both analytes. The compounds were extracted using protein precipitation coupled with liquid-liquid extraction with flash freezing technique. Negative mass transitions (m/z) of acitretin, isoacitretin and acitretin-d3 were detected in multiple reactions monitoring (MRM) mode at 325.4 → 266.3, 325.2 → 266.1 and 328.3 → 266.3, respectively, with a turbo ion spray interface. The chromatographic separation was achieved on an Ascentis-RP amide column (4.6 × 150 mm, 5 µm) with mobile phase delivered in isocratic mode. The method was validated over a concentration range of 1.025-753.217 ng/mL for acitretin and 0.394-289.234 ng/mL for isoacitretin with a limit of quantification of 1.025 and 0.394 ng/mL. The intra-day and inter-day precisions were below 8.1% for acitretin and below 13.8% for isoacitretin, while accuracy was within ±7.0 and ±10.6% respectively. For the first time, the best possible conditions for plasma stability of acitretin and isoacitretin are presented and discussed with application to clinical samples.

Rational design for variability minimization in bioanalytical method validation: illustration with LC-MS/MS assay method for terbinafine estimation in human plasma.

Terbinafine, a widely used antifungal drug, is a challenging molecule for quantitative bioanalysis due to certain factors contributing assay variability. Despite previous attempts at human plasma determination of terbinafine, exhaustive stability of the drug or an internal standard was lacking. Internal standard stability with negligible variation throughout the analysis is an indicator of a reliable bioanalytical method as the majority of LC-MS/MS assays are based on analyte/IS response ratios for quantitation. A newly developed high-throughput simple LC-MS/MS method is described for human plasma determination of terbinafine using naftifine internal standard and eluting all compounds within 2 min. A solid-phase extraction of terbinafine achieving mean recovery of 84.3% (CV < 4%) without compromising sensitivity (limit of quantitation 5.11 ng/mL) or linearity (5.11-3014.19 ng/mL) is delineated in this paper. A heated nebulizer in positive multiple reaction monitoring mode was employed with transitions m/z 292.2 →141.1 and 288.2 →117.0 for terbinafine and naftifine, respectively, resulting in excellent chromatographic separation on a Hypurity Advance (50 x 4.6 mm, 5 µm) column. The developed method was successfully applied to clinical samples and for the first time demonstrated marked improved extraction efficiency and reliable long-term plasma stability results without any internal standard response variation during the entire course of study.

LC-APCI mass spectrometric method development and validation for the determination of atovaquone in human plasma.

A newly developed LC-APCI mass spectrometric method is described for human plasma determination of atovaquone using lapachol internal standard. A single-step protein precipitation technique for plasma extraction of atovaquone achieving mean recovery of 94.17% (CV 8%) without compromising sensitivity (limit of quantitation 50.3 ng/mL) or linearity (50.3 ng/mL-23924.6 ng/mL) is delineated in this paper. Heated nebulizer in negative multiple reaction monitoring mode was employed with transitions m/z 365.2 --> m/z 337.1 and m/z 240.9 --> m/z 185.7 for atovaquone and lapachol respectively in this liquid chromatographic-tandem mass spectrometric method. Excellent chromatographic separation on a Synergi 4 micro Polar-RP 80A (150 x 2.0 mm) column, using 100 microL of plasma extraction volume along with 10 microL of injection load, completing analysis run-time within 2.5 min, highlights this simple yet unique bioanalytical method. The developed method can be successfully applied to pharmacokinetic studies on atovaquone suspension administered in healthy volunteers or HIV-infected patients. Moreover full method validation results not published before are presented and discussed in detail for the first time in this article.

Bioanalytical LC-MS/MS method validation for plasma determination of topiramate in healthy Indian volunteers.

A LC-MS/MS method for plasma topiramate analysis is delineated involving least number of healthy volunteers. Topiramate and amlodipine internal standard (IS) were extracted by simple centrifuge-coupled solid-phase extraction and reverse-phase chromatographic separation was performed on an Ascentis C(18) column. Turbo-spray negative-ion mode multiple-reaction monitoring was selected for mass pair detection at m/z 338.3 --> 78.0 and m/z 407.3 --> 295.5 for analyte and IS respectively. The method showed a dynamic linearity range from 10.4 to 2045.0 ng/mL, lower limit of quantitation achieved at 10.4 ng/mL and finally a mass spectrometric total run time of within 2.5 min for human sample analysis. Bioequivalence was assessed successfully using this fully validated method on 16 fasted Indian male subjects with 25 mg topiramate tablet administration. An appropriate study design describes plasma samples collection up to 216 h post dose in two periods, separated by a 28 day washout period. The challenge of half-life matching for test and reference drug was achieved with 73.43 +/- 9.68 and 73.06 +/- 14.03 h, respectively, and intra-subject coefficient of variation achieved within 11% for AUCs and C(max) evaluated by non-compartmental pharmacokinetic analysis. The results of LCMS topiramate complete method validation supported by pharmacokinetic study have not been published before, and are presented and discussed for the first time in this article.