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 »  Abstract
 » Introduction
 »  Materials and Me...
 » Results
 »  Discussion and C...
 »  References
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 Table of Contents    
RESEARCH ARTICLE
Year : 2019  |  Volume : 51  |  Issue : 1  |  Page : 40-44
 

Development and clinical evaluation of a dried urine spot method for detection of morphine among opioid users


National Drug Dependence Treatment Centre, All India Institute of Medical Sciences, New Delhi, India

Date of Submission25-Jul-2018
Date of Acceptance04-Mar-2019
Date of Web Publication19-Mar-2019

Correspondence Address:
Prof. Raka Jain
Room No. 4090, 4th Floor, Teaching Block, All India Institute of Medical Sciences, Ansari Nagar, New Delhi - 110 029
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijp.IJP_305_18

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 » Abstract 


BACKGROUND: In recent years, drug testing in body fluids has gained popularity for validating self-reported drug use. The storage and transportation of urine specimens is a major concern for remote areas where the facilities for performing drug abuse testing are lacking.
AIMS AND OBJECTIVES: The aim of the present study was to develop an efficient method for testing opiate in dried urine spots (DUS) and to evaluate its clinical applicability.
MATERIALS AND METHODS: The methodology involved optimization of conditions for extraction, recovery, short-, and long-term stability (room temperature, 4°C,−20°C) for detection of opiate from dried urine spots. Further, the extraction efficiency from dried urine spots was compared with the conventional drug testing methodology. The screening was done by using enzyme-linked immunosorbent assay technique, and confirmation was achieved by gas chromatography equipped with nitrogen phosphorus detector.
RESULTS: Deionized water was found to be a suitable extracting solvent compare to bi-carbonate buffer (pH 9.2) and saline. Primary screening was achieved by 2 punches taken from a 20-μl (diameter 1.3 cm) spotted urine samples, whereas confirmation was achieved by 2 complete circles each of 20 μl sample volume. The recovery was found to be 99.41% in water. No sign of significant degradation was seen among all storage conditions.
CONCLUSIONS: In the current study, DUS has achieved the same level of precision and reproducibility as that of standard methods used for drug testing in urine. Hence, the DUS sampling appears to have potential to detect opiate among drug users in a clinical setting.


Keywords: Dried urine spots, drug abuse screening, gas chromatography, immunoassay, morphine


How to cite this article:
Jain R, Quraishi R, Verma A, Ambekar A. Development and clinical evaluation of a dried urine spot method for detection of morphine among opioid users. Indian J Pharmacol 2019;51:40-4

How to cite this URL:
Jain R, Quraishi R, Verma A, Ambekar A. Development and clinical evaluation of a dried urine spot method for detection of morphine among opioid users. Indian J Pharmacol [serial online] 2019 [cited 2019 Jul 21];51:40-4. Available from: http://www.ijp-online.com/text.asp?2019/51/1/40/254583





 » Introduction Top


Opioids top the list of “problem” drugs worldwide.[1] The use of illicit opioids like heroin along with nonmedical use of prescription opioids is a rising public health concern. This leads to increased healthcare services and deaths due to drug overdose. Among injecting drug users (IDUs), the use of opioids, especially heroin is emerging. There are an estimated 15.9 million IDUs globally. In India, the number of IDUs is around 1.77 million.[2] Notably, almost all the IDUs in India use opioid drugs and are mostly opioid dependent.[3]

Laboratory screening of substance use, especially opioids to monitor drug use is an important component of addiction treatment settings. It provides an unbiased, reproducible, and accurate method to monitor patients and offers objective support for clinical considerations. Urine is often preferred over other biological specimen, as it is readily collectible and has higher levels of drugs and metabolites in comparison to serum/blood or saliva. Drug abuse testing in patients belonging to remote areas is considered to be fraught with many logistic problems. In addition, to minimize variations, it is always advisable for the analysis to be carried out in one centralized laboratory. Transportation to a distant laboratory often involves challenges such as requirement of trained staff, sample spillage, breakage, cross contamination, and shipment in lower temperatures.

Use of dry biological specimen such as dried blood spot (DBS) or dried urine spot (DUS) is becoming popular recently, as it offers some interesting advantages over liquid samples for screening/diagnosis.[4],[5],[6],[7],[8],[9],[10],[11],[12] First, it requires a less invasive sampling method. Second, DUS provides stability even at ambient temperature. It also offers easy storage and transportation with no need for freezers or dry ice in most applications. Concerning morphine and drugs of abuse, there is a paucity of literature regarding use of dried urine spots for screening. Some recent reports are available on using dried urine spots method for morphine screening.[13],[14],[15] However, no study has reported the use of dried urine spots in an addiction treatment setting and clinical validation for morphine screening till date.

The objective of the present study was to develop a simple and cost-effective method for direct screening of morphine using urine spotted onto a filter paper and evaluating its clinical applicability.


 » Materials and Methods Top


Chemicals

All the reagents used were of analytical grade and were obtained from Merck, USA. Morphine standard was obtained morphine injection IP 15 mg/ml, Troikaa Pharmaceuticals Ltd, Gujarat, India. Whatman Filter paper 903 was obtained from GE Health Care, India.

Instrumentation and gas chromatography conditions

Screening was performed by enzyme-linked immunosorbent assay (ELISA) technique (Tecan GENios ELISA reader, Austria GmbH, Austria) using Megellan Software V1.30 (Tecan Austria, Austria).

Further confirmation of the samples was done using gas chromatograph (model 7890A, Agilent India Pvt. Ltd, USA). The gas chromatography (GC) was equipped with 7683B series Auto Sampler (split/split-less inlet, fused silica capillary column coated with HP-5 cross-linked 5% diphenyl, and 95% dimethylpolysiloxane (30 m × 0.320 i.d., 0.5 μm film thickness). The temperature gradient used was 1 min at 170°C, 10°C/min from 170°C to 270°C, and 5 min at 270°C. Nitrogen was used as a carrier gas at a flow rate of 10 ml/min. The injector temperature was 280°C. The injection volume was 2 μl for each GC run, and the split ratio was kept 1:10. The nitrogen phosphorus detector (NPD) was used with electronic pneumatic control at 300°C. System control, data acquisition, and analysis were performed with GC Chem Station G2075BA software.

Sample collection

The study was conducted at a tertiary-care addiction treatment setting of North India. The inclusion criteria included male patients fulfilling International Classification of Diseases-10 criteria[16] for harmful or dependent opioid use, recent use, within last 48 h, and willing to participate in the study. The patients who reported use of drugs other than opioid and presence of any other psychiatric illness were excluded from the study. Patient informed consent was obtained from all participants who fulfilled the inclusion criteria. After inclusion, 30 ml of urine samples were collected from each patient. Drug-free urine samples (controls) were obtained from laboratory staff volunteers. The urine samples were screened by cassette test for opiates, benzodiazepines, cannabis, and nicotine use. The study was carried out in accordance with Declaration of Helsinki, and the study protocol was approved by the institutional ethics committee. The study was completed in 1-year duration (March 2012–2013).

DUS preparation

Control urine samples with zero baseline drug level were spiked with various concentrations (100, 50, 10, and 1 μg/ml) of standard morphine. Twenty ul of morphine standards spiked in control urine sample and patient's samples were spotted onto filter paper kept on a nonabsorbent surface. The spotted samples were then allowed to dry overnight at ambient temperature.

Extraction and detection of morphine from DUS

Optimal conditions were worked out to check the maximum recovery using three different eluting solvents at different temperature (room temperature and 37°C) and time conditions (4, 24, 48, 72, and 96 h). Briefly, DUS with spiked morphine standards was punched manually using a manual puncher of diameter 3.2 mm. Three different solvents, i.e., deionized water, sodium carbonate-bicarbonate buffer (pH 9.2), and saline were tried for morphine extraction from DUS. The detection of morphine was checked with ELISA kits (Calbiotech Pvt limited, USA) onto ELISA reader as per manufacturer's guidelines. The results were given as positive or negative based on the cutoff (100 ng/ml) of ELISA kit being used.

Confirmation of the urine and dried urine spots (DUS) were carried out as per the previously developed laboratory method by extraction and derivatization before injecting to gas chromatograph.[17]

Method validation

Percent recovery was calculated based on comparison between the standard and the extracted morphine from DUS using the following formula:



Where, Xe = Mean concentration obtained from extracted standard using DUS method, and Xs = mean concentration obtained from direct standard.

For interday precision, the standards were run in triplicate for three consecutive days, and for intraday precision, the standards were repeated three times in triplicate within the same day. Both are expressed as standard deviation and coefficient of variation (% CV).

Clinical validation

The use of filter paper to screen drug use in a clinical setting was checked by collecting urine samples from opioid using patients as per the inclusion criteria. Twenty microliters of the urine samples from each patient were spotted onto filter paper and dried. The filter paper spotted urine samples (DUS), and corresponding urine samples were analyzed for morphine as per the standardized conditions.

Stability

The stability of DUS was tested by spotting the morphine spiked urine samples (1 ug/ml, 500 ng/ml) and patient's urine samples on filter paper, which were kept in sealed plastic bags with desiccants until analysis. These sealed plastic bags were stored at three different temperatures (−20°C, 4°C, and room temperature [21°C–28°C]). Estimation was carried out at different points of time, i.e., within 24 h after collection and weekly for 1st 4 weeks and then consecutive weeks till 3 months.


 » Results Top


Optimization of DUS extraction

The extraction of morphine was carried out using three extracting solvents for different time periods. The recovery of morphine was found to be maximum (99.4%) using two punches (3.2 mm each) in 500 μL of deionized water, kept at 37°C in a water bath shaker for a period of 24 h [Figure 1].
Figure 1: Recovery curve of morphine

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Limit of detection (LOD) for the method was found to be 100 ng/ml. The interday CV and intraday CV results are shown in [Table 1].
Table 1: Interday and intraday coefficient of variation results

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Clinical validation

Urine samples were collected from 50 patients with opioid use disorders as per the inclusion criteria. All the patients were males with the mean age of 35.2 ± 9.67 years. Twenty-six percentage of the patients were unmarried (n = 13) while 74% were married (n = 37). Majority of the patients were employed (86%, n = 43) and only 14% were unemployed (n = 7). The education status was reported as illiterate 5 (10%), just literate 13 (26%), primary 10 (20%), high school 12 (24%), higher secondary 2 (4%), graduate 5 (10%), and postgraduate 3 (6%).

All the 50 samples were found to be positive for morphine in direct urine as well as filter paper-spotted urine samples by ELISA technique. Representative gas-liquid chromatography (GLC) chromatogram of morphine-spiked control urine with a retention time of 12.89 min is shown in [Figure 2].
Figure 2: Gas-liquid chromatography chromatogram of standard morphine-spiked control urine

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[Figure 3] shows the GLC chromatogram of a patient's urine sample by filter paper method with retention time of 12.89 min.
Figure 3: Gas-liquid chromatography chromatogram of a patient's urine sample by filter paper method

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The results showed 100% concordance between filter paper-extracted urine specimens and direct urine specimens in patients' sample using GC technique.

Stability

Morphine was found to be stable in DUS samples stored at room temperatures, −20°C and 4°C [Figure 4]. The concentration of morphine depicted in figure corresponds to 20 μl of urine spot extracted and estimated as per the optimized conditions. The stored spotted filter paper showed no degradation at the end of 3 months.
Figure 4: Stability of morphine using DUS method

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 » Discussion and Conclusions Top


Analysis of drug use in biological fluids plays an important role in addiction treatment services. Opioids are a class of drugs that includes heroin and other legal prescription analgesics such as oxycodone, hydrocodone, codeine, morphine, and fentanyl.[18] Urine is often the most preferred specimen among biological specimens used to screen recent drug use due to its ease of collection, higher detectable concentrations of drugs, and metabolites.

Through this study, a validated method for the qualitative analysis of morphine in small volumes of urine (20 μL/spot) spotted as DUS has been developed. The extraction and screening of morphine in DUS were found comparable to the conventional urine sampling method.

In the current study, LOD was found to be fairly sensitive (100 ng\ml) for screening the recent opioid use. The current method appeared to be fairly sensitive and cost-effective in a clinical setting.

Following the new challenges encountered in the process of drugs' determination, DUS method has the potential to overcome these challenges. However, in case of morphine detection from DUS, few published reports lack the clinical validation.[13],[14],[15] Some researchers reported use of DBS to assess the stability of morphine, which complements our results of stability.[19],[20] The current study advocates the use of DUS to improve morphine stability even when stored at room temperature (up to 3 months). This is in accordance with the published report; the use of filter paper stabilizes the analyte thus increasing the chance of getting a positive result even with low concentrations for a considerable period of time after sample collection.[13]

As per our knowledge, this is the first study to report the clinical validation of DUS in an addiction treatment setting to screen morphine.

This methodology was developed for the qualitative screening of morphine, and future studies with quantitative estimation with larger sample size are needed. Dried urine spot samples have been found to be a useful alternative for biological monitoring of morphine use, especially in developing countries where sample logistics could be a major problem.

Moreover, the DUS can be used as an alternative procedure in conjunction with conventional methods. It holds promise for preserving unstable drugs. It may help to avoid potential errors in analytical interpretation of results because of environmental influences. DUS has the potential to be used in field-based studies lacking laboratory facilities. Thus, the use of DUS for detecting morphine in the addiction treatment settings represents an inexpensive, rapid, precise, and simple method. These results call for further work for wider screening of participants belonging to drug using areas with limited laboratory facilities.

Financial support and sponsorship

This study was financially supported by Indian Council of Medical Research, Government of India.

Conflicts of interest

There are no conflicts of interest.



 
 » References Top

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World Drug Report; 2015. Available from: https://www.unodc.org/documents/wdr2015/World_Drug_Report_2015.pdf. [Last accessed on 2018 Jun 14].  Back to cited text no. 1
    
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Solomon SS, Srikrishnan AK, Mehta SH, Vasudevan CK, Murugavel KG, Thamburaj E, et al. High prevalence of HIV, HIV/hepatitis C virus coinfection, and risk behaviors among injection drug users in Chennai, India: A cause for concern. J Acquir Immune Defic Syndr 2008;49:327-32.  Back to cited text no. 2
    
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Ambekar A, Rao R, Mishra AK, Agrawal A. Type of opioids injected: Does it matter? A multicentric cross-sectional study of people who inject drugs. Drug Alcohol Rev 2015;34:97-104.  Back to cited text no. 3
    
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Quraishi R, Jain R, Ambekar A. The use of dried blood spot samples in screening drugs of abuse. Pharmacol Pharm 2013;4:152-9.   Back to cited text no. 4
    
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Alfazil AA, Anderson RA. Stability of benzodiazepines and cocaine in blood spots stored on filter paper. J Anal Toxicol 2008;32:511-5.  Back to cited text no. 5
    
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Havard G, Théberge MC, Boudreau N, Lévesque A, Massé R. Development and validation of a dried blood spot assay for the determination of midazolam in human whole blood by LC-MS-MS. Proceedings of the American Association of Pharmaceutical Scientists (aaps) – Pharmaceutical Sciences World Congress. New Orleans, LA; 2010.  Back to cited text no. 6
    
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Thomas A, Déglon J, Steimer T, Mangin P, Daali Y, Staub C, et al. On-line desorption of dried blood spots coupled to hydrophilic interaction/reversed-phase LC/MS/MS system for the simultaneous analysis of drugs and their polar metabolites. J Sep Sci 2010;33:873-9.  Back to cited text no. 7
    
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Jantos R, Skopp G. Comparison of drug analysis in whole blood and dried blood spots. Toxichem Krimtech 2011;78:268-75.  Back to cited text no. 8
    
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Langel K, Uusivirta H, Ariniemi K, Lillsunde P. Analysis of Drugs of Abuse by GC-MS in Dried Blood Spot Sample Matrix. Presented at: 49th Annual Meeting of The International Association of Forensic Toxicologists (TIAFT). San Francisco, CA, USA; 2011.  Back to cited text no. 9
    
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Lauer E, Déglon J, Versace F, Thomas A, Mangin P, Staub C. Target Screening of Drugs from Dried Blood Spot Samples Based on LC-MS/MS and On-Line Desorption. Presented at: 49th Annual Meeting of The International Association of Forensic Toxicologists (TIAFT). San Francisco, CA, USA; 2011.  Back to cited text no. 10
    
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Déglon J, Thomas A, Mangin P, Staub C. Direct analysis of dried blood spots coupled with mass spectrometry: Concepts and biomedical applications. Anal Bioanal Chem 2012b; 402:2485-98.  Back to cited text no. 11
    
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Déglon J, Versace F, Lauer E, Widmer C, Mangin P, Thomas A, et al. Rapid LC-MS/MS quantification of the major benzodiazepines and their metabolites on dried blood spots using a simple and cost-effective sample pretreatment. Bioanalysis 2012;4:1337-50.  Back to cited text no. 12
    
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DuBey IS, Caplan YH. The storage of forensic urine drug specimens as dry stains: Recovery and stability. J Forensic Sci 1996;41:845-50.  Back to cited text no. 13
    
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Otero-Fernández M, Cocho JÁ, Tabernero MJ, Bermejo AM, Bermejo-Barrera P, Moreda-Piñeiro A, et al. Direct tandem mass spectrometry for the simultaneous assay of opioids, cocaine and metabolites in dried urine spots. Anal Chim Acta 2013;784:25-32.  Back to cited text no. 15
    
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World Health Organization. ICD-10 Classifications of Mental and Behavioural Disorder: Clinical Descriptions and Disgnostic Guidelines. Geneva: World Health Organization; 1992.  Back to cited text no. 16
    
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Clavijo CF, Hoffman KL, Thomas JJ, Carvalho B, Chu LF, Drover DR, et al. Asensitive assay for the quantification of morphine and its active metabolites in human plasma and dried blood spots using high-performance liquid chromatography-tandem mass spectrometry. Anal Bioanal Chem 2011;400:715-28.  Back to cited text no. 19
    
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Garcia Boy R, Henseler J, Mattern R, Skopp G. Determination of morphine and 6-acetylmorphine in blood with use of dried blood spots. Ther Drug Monit 2008;30:733-9.  Back to cited text no. 20
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1]



 

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