Journal of Pharmaceutical Analysis

2018, 8(6): 封3-封4.

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Abstract:

Development and validation of an HPLC-UV method for analysis of methylphenidate hydrochloride and loxapine succinate in an activated carbon disposal system

Pooja Bakshi, Andrew Korey, William Fowler, Ajay K. Banga

2018, 8(6): 349-356.

Abstract(90) PDF(4)

Abstract:
Unused medications have the possibility of being abused, causing serious harm to individuals who were not prescribed the drug. The Food and Drug Administration (FDA) recommends the proper disposal of unused prescribed medications to maintain safety and prevent environmental hazards. However, many of the current disposal techniques do not properly address safety. A drug disposal pouch containing granular activated carbon offers a unique disposal method to deactivate residual or expiredmedication in a convenient, effective, and safe manner. A robust and validated method for methylphenidate hydrochloride and loxapine succinate was developed using high-performance liquid chromatography (HPLC) and the deactivation efficiency of the disposal system was tested. Methylphenidate hydrochloride was analyzed on a C18 analytical column (250mm × 4.60mm, 100?) using acetonitrile-water (0.05% (v/v) trifluoroacetic acid) as the mobile phase at a flow rate of 1.0mL/min with a run time of 15min and retention time of 7.8min. Loxapine succinate was separated on a C8100? (250 mm × 4.6 mm, 5 μm) column maintained at 25 °C using a flow rate of 1.0mL/min. The run time was 10min and the retention time of the drug was around 4.6min.Mobile phase was composed of acetonitrile and water (0.3% triethylamine) at pH 3.0 as 40:60 (v/v). Reference standard solutions (100 μg/mL) for both drugs were prepared by dissolving in mobile phases. These methods provide good linearity (R2 = 0.999) over the range of 5–100 μg/mL for methylphenidate hydrochloride and 0.1–100 μg/mL for loxapine succinate. The assay methods were successfully applied to study the deactivation of these drugs.

LC and LC–MS/MS studies for the identification and characterization of degradation products of acebutolol

Uday Rakibe, Ravi Tiwari, Anand Mahajan, Vipul Rane, Pravin Wakte

2018, 8(6): 357-365.

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Abstract:
The aim of the present investigation was to demonstrate an approach involving use of liquid chromatography (LC) and liquid chromatography-mass spectrometry (LC–MS) to separate, identify and characterize very small quantities of degradation products (DPs) of acebutolol without their isolation from the reaction mixtures. The drug was subjected to oxidative, hydrolytic, thermal and photolytic stress conditions as per International Conference on Harmonization (ICH) guideline Q1A(R2). Among all the stress conditions the drug was found to be labile in hydrolytic (acidic & basic) and photolytic stress conditions, while it was stable in water-induced hydrolysis, oxidative and thermal stress conditions. A total of four degradation products were formed. A C18 column was employed for the separation of all the DPs on a gradient mode by using high-performance liquid chromatography (HPLC). All the DPs were characterized with the help of their fragmentation pattern and the masses obtained upon LC–MS/MS and MSn analysis. All the hitherto unknown degradation products were identified as 1-(2-(2-hydroxy-3- (isopropylamino)propoxy)-5-(amino)phenyl)ethanone (DP-I), N-(4-(2-hydroxy-3-(isopropylamino) propoxy)-3-acetylphenyl)acrylamide (DP-II), 1-(2-(2-hydroxy-3-(isopropylamino)propoxy)-5-(hydroxymethylamino) phenyl)ethanone (DP-III) and 1-(6-(2-hydroxy-3-(isopropylamino)propoxy)-2,3-dihydro- 2-propylbenzo[d]oxazol-5-yl)ethanone (DP-IV). Finally the in-silico carcinogenicity and hepatotoxicity predictions of the drug and all the DPs were performed by using toxicity prediction softwares viz., TOPKAT, LAZAR and Discovery Studio ADMET. The results of in-silico toxicity studies revealed that acebutolol (0.967) and DP-I (0.986) were found to be carcinogenic, while acebutolol (0.490) and DP-IV (0.437) were found to be hepatotoxic.

Detection and determination of undeclared synthetic caffeine in weight loss formulations using HPLC-DAD and UHPLC-MS/MS

Carine Viana, Gabriela M. Zemolin, Thaís R. Dal Molin, Luciana Gobo, Sandra Maria Ribeiro, Gabriela C. Leal, Gabriela Z. Marcon, Leandro M. de Carvalho

2018, 8(6): 366-372.

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Caffeine is present in products marketed for weight loss, with the purpose of increasing thermogenesis and lipid metabolism. The dosage declared by the product manufacturer, or even its presence, is not always correctly described on the label. This work aimed to investigate the undeclared synthetic caffeine in weight loss formulations by a high-performance liquid chromatography with diode array detection (HPLC-DAD) method. From one hundred products purchased through Brazilian e-commerce, seventeen contained caffeine, either naturally or synthetically added to formulation. The caffeine-containing samples were confirmed by an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method, and adulteration was clearly proven in five products. The content highest caffeine contained 448.8 mg per dose. Other irregularities were also found; nevertheless, the most serious was the addition of synthetic drugs without asking the consumers. Additional drugs expose the consumer to more possible side-effects as well as deleterious drug interactions. Intentional adulteration with any unlabeled substance is typically motivated by a desire to increase or alter the claimed effect of the marketed product to gain a commercial advantage.

Gas chromatography-mass spectrometry method for determination of β-propiolactone in human inactivated rabies vaccine and its hydrolysis analysis

Shuo Lei, Xun Gao, Yang Sun, Xiangyong Yu, Longshan Zhao

2018, 8(6): 373-377.

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A simple method was established for the determination of β-propiolactone (BPL) in human inactivated rabies vaccine by gas chromatography-mass spectrometry (GC-MS). The determination was performed on an Agilent HP-INNOWAX (30m × 0.32mm i.d., 0.25 μm) capillary column at the temperature of 80 °C. Electrospray ionization (ESI) was used by selective ion detection at m/z 42. The temperature for ESI source and inlet was set at 230 °C and 200 °C, respectively. Helium was used as the carrier gas at a flow rate of 25.1 mL/min. The total run time was 8 min. Acetonitrile and other components in the sample did not interfere with the determination of BPL. The results showed good linearity of BPL in the range of 0.50–10.01 μg/mL, with the limit of detection and the limit of quantification of 0.015 μg/mL and 0.050 μg/mL, respectively. Satisfactory precision was achieved for the current developed method. The method was applied to detect 6 batches of vaccine samples, and the results indicated that the target analyte BPL was present in three batches of unpurified samples, but was not detected in the purified samples, indicating the test samples were qualified. The established method was proved to be simple, versatile and sensitive, which can meet the requirements of quality control of BPL in human inactivated rabies vaccine.

Highly sensitive LC–MS/MS method to estimate doxepin and its metabolite nordoxepin in human plasma for a bioequivalence study

Nirav P. Patel, Mallika Sanyal, Naveen Sharma, Dinesh S. Patel, Pranav S. Shrivastav, Bhavin N. Patel

2018, 8(6): 378-385.

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Abstract:
A selective, sensitive and rugged liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay has been developed for the simultaneous determination of doxepin (Dox) and its pharmacologically active metabolite, nordoxepin (NDox) in human plasma. The analytes and their internal standards (IS) were extracted from 500 μL of human plasma by liquid-liquid extraction using methyl tert-butyl ether. Chromatographic separation was achieved on Hypurity C8 column (100 mm × 4.6 mm, 5 μm) using a mixture of acetonitrile-methanol (95:5, v/v) and 2.0 mM ammonium formate in 93:7 (v/v) ratio. Detection was accomplished by tandem mass spectrometry in the positive ionization and multiple reaction monitoring acquisition mode. The protonated precursor to product ion transitions studied for Dox, NDox, and their corresponding ISs, propranolol and desipramine, were m/z 280.1-107.0, 266.0 -107.0, 260.1-116.1 and 267.1-72.1, respectively. A linear dynamic range of 15.0–3900 pg/mL for Dox and 5.00– 1300 pg/mL for NDox was established with mean correlation coefficient (r2) of 0.9991 and 0.9993, respectively. The extraction recovery ranged from 86.6%–90.4% and 88.0%–99.1% for Dox and NDox, respectively. The intra-batch and inter-batch precision (% CV) across quality control levels was ≤ 8.3% for both the analytes. Stability evaluated under different storage conditions showed no evidence of degradation and the % change in stability samples compared to nominal concentration ranged from 4.7% to 12.3%. The method was successfully applied to a bioequivalence study of 6 mg doxepin hydrochloride orally disintegrating tablet in 41 healthy Indian subjects under fasting and fed conditions.

Long-term stability of gentamicin sulfate-ethylenediaminetetraacetic acid disodium salt (EDTA-Na2) solution for catheter locks

Anne-Sophie Fiolet, Elise Jandot, Pauline Doucey, Coralie Crétet, Célia Brunel, Christine Pivot, Jean-Marc Ghigo, Christophe Beloin, David Lebeaux, Fabrice Pirot

2018, 8(6): 386-393.

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A lock solution composed of gentamicin sulfate (5 mg/mL) and ethylenediaminetetraacetic acid disodium salt (EDTA-Na2, 30 mg/mL) could fully eradicate in vivo bacterial biofilms in totally implantable venous access ports (TIVAP). In this study, fabrication, conditioning and sterilization processes of antimicrobial lock solution (ALS) were detailed and completed by a stability study. Stability of ALS was conducted for 12 months in vial (25 °C ± 2 °C, 60% ± 5% relative humidity (RH), and at 40 °C ± 2 °C, RH 75% ± 5%) and for 24 h and 72 h in TIVAP (40 °C ± 2 °C, RH 75% ± 5%). A stability indicating HPLC assay with UV detection for simultaneous quantification of gentamicin sulfate and EDTA-Na2 was developed. ALS was assayed by ion-pairing high performance liquid chromatography (HPLC) needing gentamicin derivatization, EDTA-Na2 metallocomplexation of samples and gradient mobile phase. HPLC methods to separate four gentamicin components and EDTA-Na2 were validated. Efficiency of sterility procedure and conditioning of ALS was confirmed by bacterial endotoxins and sterility tests. Physicochemical stability of ALS was determined by visual inspection, osmolality, pH, and sub-visible particle counting. Results confirmed that the stability of ALS in vials was maintained for 12 months and 24 h and 72 h in TIVAP.

Cytotoxic effect of Rosa canina extract on human colon cancer cells through repression of telomerase expression

Ibrahim Turan, Selim Demir, Kagan Kilinc, Serap Ozer Yaman, Sema Misir, Hanife Kara, Berna Genc, Ahmet Mentese, Yuksel Aliyazicioglu, Orhan Deger

2018, 8(6): 394-399.

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Rosa canina is a member of the genus Rosa that has long been used for medical objectives. Several studies have reported cytotoxic effects of different Rosa species, but there has been only limited investigation of the cytotoxic effect of R. canina. The purpose of the current study was to examine the potential effect of R. canina extract on cell viability, the cell cycle, apoptosis, and the expression of telomerase in human colon cancer (WiDr) cells. The cytotoxic effect of the extract was determined using MTT assay. The mechanism involved in the cytotoxic effect of the extract was then evaluated in terms of apoptosis and the cell cycle using flow cytometry. Mitochondrial membrane potential (MMP) was investigated using the fluorometric method, and expression levels of telomerase were studied using RT-PCR. R. canina extract exhibited a selective cytotoxic effect on WiDr cells compared with normal colon cells. The extract induced cell cycle arrest at the S phase and apoptosis via reduced MMP in WiDr cells. R. canina extract significantly repressed telomerase expressions at treatment times of 48 and 72 h in WiDr cells. Our results suggest that R. canina may have considerable potential for development as a novel natural product-based anticancer agent.

Evaluation of naproxen-induced oxidative stress, hepatotoxicity and in-vivo genotoxicity in male Wistar rats

Mir Hilal Ahmad, Mahino Fatima, Mobarak Hossain, Amal Chandra Mondal

2018, 8(6): 400-406.

Abstract(124) PDF(1)

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Naproxen (NP), a nonsteroidal anti-inflammatory drug (NSAID), is used for the treatment of common pain, inflammation and tissue damage. Genotoxicity testing of NP is of prime importance as it represents the largest group of drugs to which humans are exposed. Not many genotoxic studies are reported on NP; therefore, the present study investigated the detailed genotoxic and oxidative stress properties of NP. Male Wistar rats were administered NP orally at the doses of 38.91 and 65.78 mg/kg body weight for 14 days. Reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and lipid peroxidation (LPO) activities/levels were measured in the liver, kidney and brain tissues. The aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) activities, and total bilirubin (TBIL) levels were measured in the liver tissues. Micronucleus frequency (micronucleus test MNT) and DNA damage (comet assay) were performed in the bone marrow cells and leukocytes, respectively. The results showed that NP treatment decreased the GSH levels and increased the SOD, CAT, LPO, ALT, AST, ALP and TBIL activities/levels compared to the control (p < 0.05). Results of MNT showed an increased micronucleus induction and comet assay showed a significant increase in DNA damage in the NP treated animals (p < 0.05). Treatment of NP resulted in the biochemical imbalance and induced oxidative stress that deteriorated the integrity of the cells, which caused significant damage to the genetic material and affected liver function in male Wistar rats. Therefore, NP is a potential genotoxic agent that induces genotoxicity and oxidative stress.

Recovery rates of combination antibiotic therapy using in vitro microdialysis simulating in vivo conditions

Jayesh A. Dhanani, Suzanne L. Parker, Jeffrey Lipman, Steven C. Wallis, Jeremy Cohen, John Fraser, Adrian Barnett, Michelle Chew, Jason A. Roberts

2018, 8(6): 407-412.

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Microdialysis is a technique used to measure the unbound antibiotic concentration in the interstitial spaces, the target site of action. In vitro recovery studies are essential to calibrating the microdialysis system for in vivo studies. The effect of a combination of antibiotics on recovery into microdialysate requires investigation. In vitro microdialysis recovery studies were conducted on a combination of vancomycin and tobramycin, in a simulated in vivo model. Comparison was made between recoveries for three different concentrations and three different perfusate flow rates. The overall relative recovery for vancomycinwas lower than that of tobramycin. For tobramycin, a concentration of 20μg/mL and flow rate of 1.0μL/min had the best recovery. A concentration of 5.0μg/mL and flow rate of 1.0μL/min yielded maximal recovery for vancomycin. Large molecular size and higher protein binding resulted in lower relative recoveries for vancomycin. Perfusate flow rates and drug concentrations affected the relative recovery when a combination of vancomycin and tobramycin was tested. Low perfusate flow rates were associated with higher recovery rates. For combination antibiotic measurement which includes agents that are highly protein bound, in vitro studies performed prior to in vivo studies may ensure the reliable measurement of unbound concentrations.

Novel ligand-based docking; molecular dynamic simulations; and absorption, distribution, metabolism, and excretion approach to analyzing potential acetylcholinesterase inhibitors for Alzheimer's disease

Subramaniyan Vijayakumar, Palani Manogar, Srinivasan Prabhu, Ram Avadhar Sanjeevkumar Singh

2018, 8(6): 413-420.

Abstract(127) PDF(3)

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Acetylcholinesterase (AChE) plays an important role in Alzheimer's disease (AD). The excessive activity of AChE causes various neuronal problems, particularly dementia and neuronal cell deaths. Generally, anti- AChE drugs induce some serious neuronal side effects in humans. Therefore, this study sought to identify alternative drug molecules from natural products with fewer side effects than those of conventional drugs for treating AD. To achieve this, we developed computational methods for predicting drug and target binding affinities using the Schrodinger suite. The target and ligand molecules were retrieved from established databases. The target enzyme has 539 amino acid residues in its sequence alignment. Ligand molecules of 20 bioactive molecules were obtained from different kinds of plants, after which we performed critical analyses such as molecular docking; molecular dynamic (MD) simulations; and absorption, distribution, metabolism, and excretion (ADME) analysis. In the docking studies, the natural compound rutin showed a superior docking score of -12.335 with a good binding energy value of -73.313 kcal/mol. Based on these findings, rutin and the target complex was used to perform MD simulations to analyze rutin stability at 30 ns. In conclusion, our study demonstrates that rutin is a superior drug candidate for AD. Therefore, we propose that this molecule is worth further investigation using in vitro studies.

2018 Vol. 8, No. 6