Development and validation of LC-MS/MS method for determination of non-opioid analgesics in adulterated herbal medicines
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Date
2022-11
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Sokoine University of Agriculture
Abstract
Background: Herbal medicines are increasingly used worldwide despite the
increased concern of their adulteration with conventional drugs. Non-opioid
analgesics are among of the conventional drugs reported to be used in
adulteration of anti-pain herbal medicines. This rise a need for developing
reliable analytical method for determination of adulterated conventional
drugs in herbal medicines for quality control. This study aimed at developing
a high throughput Liquid Chromatography coupled with Tandem Mass
Spectrometry for determination of non-opioid analgesics (acetaminophen,
caffeine, acetylsalicylic acid, diclofenac, and ibuprofen) in herbal medicines
in one run. The study also aimed at optimizing a sample preparation
technique to enhance detection of the adulterants in herbal medicine
obtained from the Tanzania market.
Methodology: Solid Phase Extraction (SPE) and Ultrasonic Assisted
Extraction (UAE) methods were compared in respect of recoveries,
extraction time, complexity, matrix effects, and expenses for sample
preparation. Waters OASIS Hydrophilic-lipophilic balance (HLB) 200 mg 6
mL, a C 18 sorbent was used in Solid Phase Extraction method optimization.
The optimized parameters were effects of filters on sample filtration, sample
loading pH, selection of suitable solvents for conditioning, washing, and
elution. Ultrasonic bath and centrifuge were used for optimization of
ultrasonic assisted extraction method parameters including effectives of
organic solvent in extraction, extraction solvent content, and sample to
extraction solvent volume ratio. Both methods were evaluated by calculating
their matrix effects, absolute recoveries, and percentage recovery for sub-
steps. The effective sample preparation method was used in development
and validation of an advanced hyphenated method using an Agilent
Technologies 1100 series Liquid Chromatography coupled with Waters
Tandem Mass spectrometry (LC-MS/MS) with electrospray ionization. The
LC-MS/MS method was validated for its selectivity, sensitivity, linearity,
accuracy, precision, recovery, matrix effects, and stability. It was then used
to analyse 132 samples collected from the markets of four regions in
Tanzania.
Results and Discussion: The Ultrasonic Assisted Extraction method
showed to be efficient to Solid Phase Extraction method for extraction of
selected non-opioid analgesics. This was clearly evidenced by high
recoveries, minimum extraction time, good peak shape, its simplicity in
procedures, and use of less expensive consumables. The absolute
recoveries for Ultrasonic Assisted Extraction were at acceptable range (> 60%) ranging from 60 % to 107 % while for Solid Phase Extraction method
showed poor recovery except for diclofenac and ibuprofen having 83% and 67%, respectively. The matrix
effect expressed as signal suppression/enhancement ranged from 70% -
122% for Ultrasonic Assisted Extraction method and 3% to 124% for Solid
Phase Extraction method. From the results the Ultrasonic Assisted
Extraction method was chosen for sample extraction for analysis of Herbal
Samples.
The developed LC-MS/MS method was linear with coefficient of
determination of R 2 ≥ 0.9931. The Limit of Detection (LOD) and Limit of
Quantification (LOQ) for selected non-opioid analgesics were within the
range of 0 – 3.7 μg/mL. The intra-precision of the LC-MS/MS method was
expressed as Relative Standard deviation and was less than 9.5% with
varying matrix effect among analytes. Twenty one percent (21%) of 132
collected herbal medicines were adulterated with caffeine and acetylsalicylic
acid. 1,029.22 μg/mL was one of the highest concentration of caffeine
detected in one of the analysed herbal medicines.
Conclusion and recommendation: This is the first study in Tanzania to
report an analytical method for analysis of five (5) non opioid analgesics in
herbal medicines in Tanzania. The developed LC-MS/MS method is suitable
for the identification and quantification of 5 non opioid analgesics explored
adulterants. The method can be adopted by the regulatory authorities for
routine analysis of herbal medicines for monitoring its quality and safety.
Further studies are recommended for Ultrasonic Assisted Extraction method
using additional internal standards.
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Keywords
Herbal medicines, Adulteration, Analgesics, Solid Phase extraction, Ultrasonic assisted extraction, LC-MS/MS