Journal of Pharmaceutical and Biomedical Analysis 129 (2016) 419–426 Contents lists available at ScienceDirect Journal of Pharmaceutical and Biomedical Analysis j o ur na l ho mepage: www.elsevier.com/locate/jpba Quantitative determination of multi markers in five varieties of Withania somnifera using ultra-high performance liquid chromatography with hybrid triple quadrupole linear ion trap mass spectrometer combined with multivariate analysis: Application to pharmaceutical dosage forms Preeti Chandra a,b , Rekha Kannujia c , Ankita Saxena d , Mukesh Srivastava e , Lal Bahadur c , Mahesh Pal d , Bhim Pratap Singh f , Sanjeev Kumar Ojha g , Brijesh Kumar a,b,∗ a Professor (AcSIR) & Senior Principal Scientist, Sophisticated Analytical Instrument Facility, CSIR-Central Drug Research Institute, Lucknow 226031, Uttar Pradesh, India b Academy of Scientific and Innovative Research (AcSIR), New Delhi 110025, India c Soil Science Division, CSIR-National Botanical Research Institute, Lucknow 226001, Uttar Pradesh, India d Phytochemistry Division, CSIR-National Botanical Research Institute, Lucknow 226001, Uttar Pradesh, India e Biometry and Statistics Division, CSIR-Central Drug Research Institute, Lucknow 226031, Uttar Pradesh, India f Department of Biotechnology, Mizoram University, Tanharil, Aizawl, Mizoram 796004, India g Ethnopharmacology Division, National Botanical Research Institute, Lucknow, Uttar Pradesh, India a r t i c l e i n f o Article history: Received 1 November 2015 Received in revised form 18 July 2016 Accepted 19 July 2016 Available online 20 July 2016 Keywords: Multiple reaction monitoring Marker quantification UHPLC–ESI-MS/MS Marketed formulation Hierarchical cluster analysis Principal component analysis a b s t r a c t An ultra-high performance liquid chromatography electrospray ionization tandem mass spectrometry method has been developed and validated for simultaneous quantification of six major bioactive com- pounds in five varieties of Withania somnifera in various plant parts (leaf, stem and root). The analysis was accomplished on Waters ACQUITY UPLC BEH C 18 column with linear gradient elution of water/formic acid (0.1%) and acetonitrile at a flow rate of 0.3 mL min −1 . The proposed method was validated with accept- able linearity (r 2 , 0.9989–0.9998), precision (RSD, 0.16–2.01%), stability (RSD, 1.04–1.62%) and recovery (RSD ≤2.45%), under optimum conditions. The method was also successfully applied for the simultane- ous determination of six marker compounds in twenty-six marketed formulations. Hierarchical cluster analysis and principal component analysis were applied to discriminate these twenty-six batches based on characteristics of the bioactive compounds. The results indicated that this method is advance, rapid, sensitive and suitable to reveal the quality of Withania somnifera and also capable of performing quality evaluation of polyherbal formulations having similar markers/raw herbs. © 2016 Elsevier B.V. All rights reserved. 1. Introduction Withania somnifera (L.) Dunal (Solanaceae) is greatly reputed medicinal plant used in Indian Ayurveda and commonly known as Ashwagandha/winter cherry/Indian ginseng [1–3]. It is used in the forms of decoctions, infusions, ointments, powder, and syrups. Withania somnifera (W. somnifera) is located mainly in arid zones of Afghanistan, Baluchistan, Congo, Jordan, Morocco, Pakistan, Sri Lanka, South Africa, and India. In India, it is widely ∗ Corresponding author at: Sophisticated Analytical Instrument Facility, CSIR- Central Drug Research Institute, Lucknow 226031, Uttar Pradesh, India. E-mail address: brijesh kumar@cdri.res.in (B. Kumar). grown in the states of Gujarat, Madhya Pradesh, Punjab, Uttar Pradesh and Rajasthan. W. somnifera has several therapeutic activ- ities, e.g. antioxidant, antimicrobial, antifungal [4], adaptogenic [5], cardioprotective [6], anticancer [7], neuroprotective, anticon- vulsant [3,8], immunomodulatory [9], apoptic [10], diuretic [11], hepatoprotective [12] and anti-inflammatory [13,14]. These ther- apeutic activities are attributed mainly due to the presence of steroidal lactones i.e., withanolides present in W. somnifera [1]. In India cultivable land is declining day by day due to various developmental activities. There are no possibilities of substitut- ing food crops with medicinal and aromatic plants in fertile land, due to increasing food requirements. In this case, the only option left is growing of medicinal and aromatic plants in salt affected http://dx.doi.org/10.1016/j.jpba.2016.07.032 0731-7085/© 2016 Elsevier B.V. All rights reserved.