Buletin SK 7(1)(2023): 20 – 23 ISSN 2250-1852
http://myjms.mohe.gov.my/index.php/bsk
Copyright © 2023 Faculty of Health Science UKM. All right reserved Artikel Asli/Original Article
Stability of Extracted Morphine from Human Urine Samples At 4C for 7 Days
Kestabilan Morfin yang Diekstrak dari Sampel Urin Manusia pada Suhu 4 C Selama 7 Hari
CHAN KAM SOON, NOR’ASHIKIN OTHMAN & AHMAD ROHI GHAZALI
ABSTRACT
Morphine is known to be a classic opiate and has years of analgesic uses as well as abuse. It is crucial to provide an accurate and reliable morphine concentration for both medico-legal and clinical purposes. Stability is identified as one of the analytical factors that could affect morphine quantification. Hence, the objective of the study was to determine the stability of the extracted morphine from human urine samples. About 10 human urine samples were selected and subjected to morphine extraction. The samples were stored at 4 ⁰C for 7 days and analysed on GCMS daily. The findings showed that the concentrations from day 2 to 7 were within the acceptable difference of ± 20 % from day 1 of collection. In conclusion, the extracted morphine from the urine samples was stable when kept at 4 ⁰C for 7 days.
Keywords: Morphine, GCMS, stability, urine
ABSTRAK
Morfin dikenali sebagai opiat klasik dan mempunyai sejarah penggunaan sebagai agen analgesik dan juga penyalahgunaan. Kepekatan morfin dalam sampel urin harus diukur dengan tepat dan sangat penting untuk tujuan mediko-legal dan klinikal. Kestabilan sesuatu analit dikenalpasti sebagai salah satu faktor analitikal yang berupaya mempengaruhi kuantifikasi morfin. Maka, objektif kajian ini adalah untuk mengenalpasti kestabilan morfin yang diekstrak dari sampel urin manusia. Sebanyak 10 sampel urin manusia telah diproses dengan ujian ekstrasi morfin.
Sampel yang diekstrak disimpan pada 4 ⁰C selama 7 hari dan dianalisa dengan GCMS setiap hari. Hasil kajian menunjukkan perbezaan kepekatan dari hari 2 ke 7 adalah dalam lingkungan ± 20 % berbanding dengan hari 1.
Kesimpulan, morfin yang diekstrak dari sampel urin manusia adalah stabil jika disimpan pada 4 ⁰C selama 7 hari.
Kata kunci: Morfin, GCMS, kestabilan, urin
INTRODUCTION
Morphine and codeine are naturally occurring alkaloids. It is well known that these alkaloids are widely used for medicinal purposes as well as recreational. The presence of the parent drugs or their active metabolites are indication of intake (Zhang et al. 2013). Various biological samples such as blood, plasma, urine, saliva, hair and nail are used for drug and toxicology testing (Oliveira et al. 2016).
The use of analytical instrument such as gas chromatography mass spectrometry (GCMS) is rapidly growing for drug testing confirmation. A variety of other developed methods for determination and quantification of morphine could deploy multi-
staged sample preparations prior to GCMS analysis too for a more significant and impactful quantification results (Salvagno et al. 2017).
One of the factors that could affect the reliability of the result is stability. Stability is defined as “the chemical stability of an analyte in a given matrix under specific conditions for given time intervals”
(Shah et al. 2000). There are a few factors that have been identified to cause instability in biological samples such as the presence of certain enzymes, temperature, pH, oxidation and light exposure (Briscoe & Hage. 2009).
In our laboratory (Drug & Toxicology Laboratory, Hospital Kuala Lumpur), the extracted morphine from human urine samples are analysed
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with GCMS on the same day of the extraction. Due to some challenges of GCMS analyser breakdown, the extracted morphine from urine samples were sometimes unable to be analysed on the day of extraction. In addition, the study on the stability of the extracted morphine from urine samples at the storage of 4 ⁰C is also lacking. Hence, our study has an opportunity to evaluate the stability of extracted morphine from human urine sample at 4⁰C storage for 7 days.
MATERIALS AND METHODS
ETHICS APPROVAL
This research was approved by Medical Research &
Ethics Committee (MREC), Ministry of Health Malaysia (NMRR ID-21-02101-ATG (IIR)).
URINE SAMPLES COLLECTION
A convenient sampling technique was deployed.
About 10 human urine samples were randomly selected and each sample was aliquot into 70 tubes (each tube represented samples from day 1 to 7). The samples were named as Sample A to J. The samples were from Agensi Antidadah Kebangsaan (AADK).
The selected samples were the leftover samples after the routine analysis.
SAMPLE PROCESSING AND GCMS ANALYSIS
For the morphine extraction procedure, 1 mL urine sample was used and spiked with 6 µL 100 µg/mL internal standard (morphine-D3) for quantification.
The samples were subjected to hydrolysis by adding 100 µL 50 % (v/v) hydrochloric acid and autoclaved at 121 ⁰C for 15 minutes. After hydrolysis process, the pH was adjusted to 5 – 6 and followed with centrifugation (3000 RCF, 3 minutes). Solid phase extraction (SPE) was used to extract the morphine from urine with Positive Pressure Manifold. A mixture of ethyl acetate : isopropanol : ammonia solution (94 : 6 : 4) was used as extraction solvents.
Derivatization was applied on the extracted morphine by adding acetic anhydride and pyridine. After that, the extracted morphine samples were incubated for 15 minutes at 45 ⁰C and brought to full dryness.
About 80 – 100 µL ethyl acetate was used to reconstitute the extracted morphine and transferred into the GCMS vials. The samples were run on GCMS to get the concentration of the extracted morphine. The present study used the established and validated extraction method to extract the morphine and analysed with a validated chromatographic
technique. Each batch of analysis was run along with the new set of calibrators and quality controls to ensure the reliability of the results.
STABILITY STUDY DURATION AND STORAGE
After the urine samples had been processed according to morphine extraction procedure, the extracted morphine samples were kept at 4⁰C for 7 days. The extracted samples were run on GCMS each day until the Day 7. Each sample had 7 replicates (each replicate represented each day).
STATISTICAL ANALYSIS
The mean readings from samples of day-2 to 7 were compared to day-1 for stability evaluation and the acceptance difference was ± 20 % (Deshpande 2020).
Each sample had triplicate readings. The data were presented as mean ± SD.
RESULTS & DISCUSSION
Fig. 1 showed the concentrations of the extracted morphine for samples from each day (day 1 to day 7) with the average of the triplicates readings. Total 10 human samples were randomly selected and the morphine was extracted and analysed with GCMS.
The concentration of sample day 1 (sample A = 5220.57 ng/mL, sample B = 1734.52 ng/mL, sample C = 640.72 ng/mL, sample D = 1030.24 ng/mL, sample E = 1016.20 ng/mL, sample F = 3169.38 ng/mL, sample G = 1166.54 ng/mL, sample H = 275.38 ng/mL, sample I = 4761.25 ng/mL, sample J = 224.39 ng/mL). Table 1 showed the difference on the morphine concentration of the samples from day 2 to day 7 as compared to day 1. The extracted morphine samples were considered as stable if the reading fell in an interval within ± 20 % of the concentration of day 1 (Deshpande 2020).
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FIGURE 1 The concentrations of the extracted morphine. The results were presented as mean ± SD of triplicate readings
TABLE 1 The difference of the concentration of each day as compared to day 1
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Sample A 5220.57 -10.82 -7.67 -19.19 -11.38 -10.66 -6.67 Sample B 1734.52 -0.02 1.75 11.66 12.45 1.15 1.89 Sample C 640.72 2.86 11.74 1.28 -2.67 14.18 0.60 Sample D 1030.24 -2.75 -6.09 -8.13 -10.26 -3.72 0.10 Sample E 1016.20 2.82 14.37 6.72 -5.57 4.45 -0.31 Sample F 3169.38 3.09 0.11 8.29 2.37 2.82 11.44 Sample G 1166.54 1.60 6.94 8.06 9.48 0.58 0.86 Sample H 275.38 -3.70 5.32 -3.47 5.31 -10.11 -9.69
Sample I 4761.25 1.02 -1.50 5.29 -4.32 1.84 -6.41 Sample J 224.39 -6.66 -6.55 -6.14 -14.40 -5.45 -13.34
*Readings from day-2 to 7 were compared to day-1 for stability evaluation and the acceptance difference was ± 20 %
** Day 1 showed the mean concentration of the extracted morphine in ng/mL
*** Day 2 to 7 showed the difference of the concentration compared to day 1 in %
Fig. 1 displayed a wide range of concentrations had been analysed with as low as 224.39 ng/mL and 5220.57 being the highest concentration. Results showed the present study had evaluated the stability at different level of concentrations. Overall, all the samples had shown a consistent concentration throughout the 7 days of analysis. The difference of the concentration of day 2 – 7 to day 1 were within acceptance range, ± 20 %. This showed that the extracted morphine was stable when kept in refrigerator at 4 ⁰C for 7 days. Our findings were in
good agreement with the study by Zhang et al. (2013).
In their study, the stability of the extracted morphine was evaluated by two conditions, three freeze-thaw cycles and autosampler ambient 12 hours at different concentrations (50, 200 and 1600 ng/mL). In addition, another study by Clavijo et al. (2011) also showed the similar finding with our study where the extracted morphine from plasma and dried blood spots were stable for at least 5 days at 4 C.
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CONCLUSION
In conclusion, the extracted morphine from the human urine samples were stable when kept at refrigerator at 4 ⁰C for seven days. This finding would be helpful to us so that the extracted morphine samples to be analysed on GCMS as soon as possible before the seven days storage. This was to ensure to get an accurate result prior to the degradation of the extracted morphine.
ACKNOWLEDGEMENT
We would like to thank the Director General of Health Malaysia for his permission to publish this article.
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Chan Kam Soon Nor’ashikin Othman
Drug & Toxicology Laboratory Pathology Department
Hospital Kuala Lumpur
Kementerian Kesihatan Malaysia 50586 Jalan Pahang,
Wilayah Persekutuan Kuala Lumpur Malaysia
Ahmad Rohi Ghazali
Center for Toxicology and Health Risk (CORE) Faculty of Health Sciences
Universiti Kebangsaan Malaysia 50300 Jalan Raja Muda Abdul Aziz Wilayah Persekutuan Kuala Lumpur Malaysia
Corresponding Author: Ahmad Rohi Ghazali E-mail: [email protected]
Tel: 019-2268976 Fax: 03-89215555
Received: 15 November 2022 Revised: 4 May 2023
Accepted for publication: 19 June 2023