title: Rapid, sensitive, and validated UPLC/Q-TOF-MS method for quantitative determination of vasicine in Adhatoda vasica and its in vitro culture.
abstract: BACKGROUND
  Adhatoda vasica a perennial herb has been used in Ayurvedic and Unani system of medicines since last 2000 years and has been employed for the treatment of respiratory tract ailments.
  
  
  OBJECTIVE
  To develop and validate new, rapid, and highly sensitive high throughput ultra-performance liquid chromatography/quadrupole-time-of-flight mass-spectrometry (UPLC/Q-TOF-MS) method for the quantitative estimation of vasicine in the leaves and to establish in vitro cultures of Adhatoda vasica for production of vasicine.
  
  
  METHODS
  The chromatographic separation was achieved on a Waters ACQUITY UPLC™ BEH C8 (100.0 × 2.1 mm; 1.7 μm) column packing using isocratic mobile phase consisting of acetonitrile: 20 mM ammonium acetate (90:10; v/v) in a multiple reactions monitoring mode using the transitions m/z 189.09 → 171.08 for vasicine.
  
  
  RESULTS
  The vasicine was eluted at 2.58 ± 0.05 min and established a dynamic range of linearity over the concentration range of 1-1000 ng/ml (r (2) = 0.999 ± 0.0005). The lower limit of detection and quantification was 0.68 and 1.0 ng/ml, respectively. There was no significant difference observed in the content of vasicine (0.92-1.04%w/w) among the eleven samples collected from different locations of India. The in vitro cultures developed showed that addition of extra 28 mM KNO3 and 100 mM NaCl in MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) + benzyladenine (BA) + indole acetic acid (IAA) (1 ppm each) produces faster biomass and higher amount of quinazoline alkaloids.
  
  
  CONCLUSIONS
  Rapid, efficient, and sensitive UPLC/Q-TOF-MS method was developed for the estimation of vasicine and an efficient protocol for development of in vitro cultures was proposed, which can be used at large scale for industrial production of vasicine using bioreactors.
authors.affiliation: Department of Pharmacognosy and Phytochemistry, Bioactive Natural Product Laboratory, Hamdard University, Hamdard Nagar, New Delhi, India.
authors.forename: Garg
authors.initials: G
authors.lastname: Madhukar
authors.affiliation: Department of Pharmacognosy and Phytochemistry, Bioactive Natural Product Laboratory, Hamdard University, Hamdard Nagar, New Delhi, India.
authors.forename: Ennus Tajuddin
authors.initials: ET
authors.lastname: Tamboli
authors.affiliation: Department of Pharmaceutics, Faculty of Pharmacy, Hamdard University, Hamdard Nagar, New Delhi, India.
authors.forename: Parveen
authors.initials: P
authors.lastname: Rabea
authors.affiliation: Department of Pharmacognosy and Phytochemistry, Bioactive Natural Product Laboratory, Hamdard University, Hamdard Nagar, New Delhi, India.
authors.forename: S H
authors.initials: SH
authors.lastname: Ansari
authors.affiliation: Department of Biotechnology, Faculty of Science, Hamdard University, Hamdard Nagar, New Delhi, India.
authors.forename: M Z
authors.initials: MZ
authors.lastname: Abdin
authors.affiliation: Department of Pharmacognosy and Phytochemistry, Bioactive Natural Product Laboratory, Hamdard University, Hamdard Nagar, New Delhi, India.
authors.forename: Ahmad
authors.initials: A
authors.lastname: Sayeed
keywords: Adhatoda vasica; UPLC/Q-TOF-MS; in vitro cultures; validation; vasicine