Development and validation of lc-ms/ms method for determination of non-opioid analgesics in adulterated herbal medicines
Loading...
Date
2022
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
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.
Key words: Herbal medicines, Adulteration, Analgesics, Solid Phase
extraction, Ultrasonic assisted extraction, LC-MS/MS. Usuli wa tatizo: Ongezeko la matumizi ya dawa zitokanazo na mimea-dawa
linazidi kukua ulimwenguni licha ya wasiwasi uliopo wa kuathiriwa kwa dawa
hizo kwa kuchanganywa na dawa za kisasa. Dawa za kutuliza maumivu za
kisasa ni miongoni mwa dawa zinazoripotiwa kuongezwa katika dawa
zitokanazo na mimea dawa za kupunguza maumivu na hivyo kushusha
ubora wake. Hivyo, kuna uhitaji mkubwa wa kuunda njia ya kisayansi yenye
kuaminika kwa ajili ya uchambuzi na utambuzi wa dawa zitokanazo na
mimea dawa zilizochanganywa na dawa za kisasa ili kudhibiti ubora na
kuongeza usalama kwa watumiaji. Utafiti huu ulilenga kutengeneza njia ya
kisayansi ya uchambuzi inayotumia teknolojia ya LC-MS/MS ili kubaini aina
na kiasi cha dawa za kisasa za kutuliza maumivu (asetaminopheni, kafeini,
aspirini, diclofenaki, na ibuprofeni) zilizomo ndani ya dawa zitokanazo na
mimea dawa. Utafiti huu pia ulilenga kuboresha mbinu ya utayarishaji wa
sampuli ili kuboresha ugunduzi wa dawa za kisasa ndani ya dawa
zitokanazo na mimea dawa zinazopatikana katika soko la Tanzania.
Methodolojia: Utafiti ulilinganisha njia mbili, Solid Phase Extraction (SPE )
na Ultrasonic Assisted Extraction (UAE), kwa kuzingatia uhuishaji, muda
ambao ulitumika kuchukua kiziduo, athari za dutu za ziada, na gharama za
uandaaji wa sampuli. Teknolojia ya Waters OASIS Hydrophilic-lipophilic
balance (HLB) 200 mg 6 mL, sobanti C18 ilitumika katika mchakato wa
kuchambua dutu lengwa. Vigezo stahiki vilipatikana kutokana na athari
zilizojitokeza katika uandaaji wa sampuli. Pia teknolojia ya ultrasonic
assisted extraction ilitumika kuweka sawa mchakato wa kikemikali wa
kuchambua dutu lengwa kutoka kwenye sampuli. Njia zote mbili
zilitathiminiwa kwa kukokotoa athari zilizojitokeza, kiasi cha dutu
zilizopatikana katika mchakato, na kiasi cha dutu kilichopatikana katika
hatua zilizofuata. Njia bora ya kuandaa sampuli stahiki ilitumika katika
kutengeneza na kuthibitisha njia yenye ufanisi wa kuchambua dutu lengwa.
Kukamilisha zoezi hili, teknolojia ya Agilent Technologies 1100 series Liquid
Chromatography na ile ya Waters Tandem Mass spectrometry (LC-MS/MS)
yenye electrospray ionization vilitumika. Njia ya LC-MS/MS ilihakikiwa uwezo
wake katika kubaini dutu husika, usahihi, utoshelevu, na umadhubuti. Aidha,
njia hii ilitumika kuchambua sampuli 132 zilizokusanywa kutoka kwenye
masoko ya mikoa minne nchini Tanzania.
Matokeo na mjadala: Njia ya UAE ilionyesha kuwa na ufanisi katika
kuandaa sampuli ya dawa za kupunguza maumivu zilizochaguliwa kwa ajili
ya utafiti. Hii ilithibitishwa na uwezo mkubwa katika kuhuisha, kutumia muda mchache, kutoa taswira nyoofu, urahisi katika kutumia pamoja na utumiaji
wa malighafi za gharama ndogo. Uhuishaji halisi wa UAE ulikuwa katika kiwango
kinachokubalika, yani, kuanzia 60 % hadi 107 %, wakati njia ya SPE
ilionesha uhuishaji dhaifu isipokuwa kwa diclofenac (83%) na ibuprofen
(67%). Athari za ziada zilizooneshwa kama alama zilizodhibitiwa (fiche)
zilianzia 70% - 122% kwa njia ya UAE na 3% hadi 124% kwa njia ya SPE.
Kutokana na matokeo, UAE ilichaguliwa kwa ajili ya kuandaa sampuli
zitokanazo na mimea dawa kwa ajili ya uchambuzi.
Njia ya LC-MS/MS ililandana na kizigeu-tambuzi, R 2 ≥ 0.9931, upeo wa
kubaini pamoja na upeo wa kiasi kwa dawa za kuzuia maumivu zilizotafitiwa
zilikuwa kati ya 0 – 3.7 μg/mL. Utashelevu wa ndani wa njia ya LC-MS/MS
ilijidhihirisha kama achano sanifu-wiano ambalo lilikuwa 9.5% iliyokuwa na
athari za ziada mbalimbali katika dutu zilizochambuliwa. Asilimia ishirini na
moja (21%) ya dawa 132 za asili zilikuwa zimechanganywa na kafeini na
aspirini. Kiasi cha juu cha kafeini kilichogunduliwa katika dawa
zilizochunguzwa ni 1,029.22 μg/mL.
Hitimisho na mapendekezo: Huu ni utafiti wa kwanza nchini Tanzania
uliotumia njia changanuzi kuchunguza uwepo wa dawa tano za kisasa ndani
ya dawa zitokanazo na mimea dawa za kutuliza maumivu. Njia iliyobuniwa
ya LC-MS/MS ilifaa katika kutambua na kupima dawa tano za kupunguza
maumivu zilizotumika kama viharibifu. Hivyo, njia hii inaweza kutumiwa na
mamlaka za udhibiti wa ubora na usalama wa dawa za asili. Inapendekezwa
kuwa tafiti zaidi zifanyike kutengeneza njia zingine za LC-MS/MS kwa dawa
zingine ili kudhibiti ubora wake.
Maneno muhimu: Dawa za mimea-dawa, kuchanganywa, dawa za kutuliza
maumivu, uziduzi, LC-MS/MS.
Description
Dissertation
Keywords
Development, Lc-ms/ms, Method, Determination, Non-opioid analgesics, Adulterated, Herbal medicines