Development and validation of lc-ms/ms method for determination of non-opioid analgesics in adulterated herbal medicines

Dissertation

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.

Danish Ministry of Foreign Affairs through Danida Fellowship Centre (DFC)