Analysis of opioids in postmortem urine samples by dispersive liquid-liquid microextraction and high performance liquid chromatography with photo diode array detection
© The Author(s) 2018
Received: 30 August 2017
Accepted: 23 January 2018
Published: 2 February 2018
Opioids abuse and related deaths are increasing in the world. Therefore, the design of new analytical methods for detection of opioids in biological samples is necessary for clinical and forensic settings.
In this study, dispersive liquid-liquid microextraction (DLLME) combined with high-performance liquid chromatography with photo diode array detector (HPLC-PDA), as a new and sensitive method were examined for the extraction and determination of morphine, codeine and methadone in postmortem urine samples. Effective factors on DLLME were optimized. The extracts were analyzed by HPLC-PDA using a Eurospher® C18 column (250 mm × 4.6 mm, particle size: 5 μm).
The volumes of chloroform as the extraction solvent and acetone as the dispersive solvent were selected 300 μl and 500 μl, respectively. The optimum pH 9.8 and extraction time was 0.5 min were selected. Under optimum condition, the enrichment factor and the recovery of morphine, codeine, and methadone spiked into postmortem urine samples were in the range of 175–215.8 and 87.5–107.9%, respectively. Calibration curves for each analyte are linear in the range of 0.5–100 μg ml− 1. Limit of detection (LOD) for the analytes was in the range of 10–25 μg l− 1. Finally, the proposed method was successfully applied to 50 postmortem urine samples for determination of the opioids.
The proposed method is an easy, fast, low cost and efficient for the extraction and determination of opioids in postmortem urine samples and should be considered as analytical method for determination of opioids in forensic and clinical toxicology labs.
In recent years, substance abuse has been widely spread in the world and has social, economic, cultural and political dimensions in the society and considered as a major health threat (UNODC 2016). Opioids are a class of analgesics commonly used in clinical medicine for treatment of moderate and sever pain (Gergov et al. 2009; Pathan and Williams 2012). Also, they have the high potential for abuse (UNODC 2016). Therefore, analysis of the opioids in biological samples has been considered as an important issue in the forensic and clinical toxicology (Gergov et al. 2009; Shamsipur and Fattahi 2011). The determination of abused drugs in postmortem samples can provide some special challenges in comparison with clinical samples (Drummer 2004). The variety and quality of the biological samples such as decomposed tissues, instability and degradation of drugs of abuse in the postmortem conditions and drug redistribution are the some special features in analysis of drugs in postmortem forensic toxicology (Drummer 2004). Furthermore, the development of new analytical methods for the qualitative and quantitative analysis of opioids in postmortem biological samples is an important concern in the forensic toxicology (Drummer 2004). A fast, easy and effective method for sample preparation is a key role for achieving to the better analytical procedures. Some traditional analytical techniques such as liquid-liquid extraction (LLE) and solid-phase extraction (SPE) have been developed for extraction and determination of drugs including opioids in biological specimens (Wey and Thorman 2001; Whittington and Kharasch 2003; Mabuchi et al. 2004). There are some limitations on using of these methods of sample preparation. LLE method is time-consuming and requires the use of large volumes of high purity and toxic organic solvents. The SPE is a method with relatively good efficacy, but it is relatively time-consuming for some long processes such as the washing and evaporation of the solvents. Also, in some cases, the method recovery is not enough for trace analysis (Shamsipur and Fattahi 2011). Therefore, the development of rapid, easy and environment– friendly analytical methods is encouraged.
Recently, microextraction procedures are the most effective sample preparation methods prior analysis. For example, liquid-phase microextraction (LPME) was successfully used for extraction of analytes from aqueous samples (Jeannot and Cantwell 1996; He and Lee 1997). Hollow fiber LPME (HF-LPME) is another easy and low-cost sample preparation method in order to extraction of analytes from complex samples (Shen and Lee 2002; Lee et al. 2008; Saraji et al. 2011). The combination of ultrasound with microextraction and solvent drop solidification (LPME-SFO) are the two examples of developed methods based on microextraction (Leong and Huang 2008; Ma et al. 2009; Cheng et al. 2011; Zhang and Lee 2012).
Dispersive liquid-liquid microextraction (DLLME) is another type of microextraction method that consists of a trinary system of solvents including a high-density and water- immiscible extraction solvent (extractant), a dispersive solvent highly miscible with the extraction solvent and aqueous sample, and an aqueous sample (Rezaee et al. 2006). The method based on the formation of very small droplets of extraction solvent in the sample solution after injection of extractant and dispersive solvent into aqueous sample (Shamsipur and Fattahi 2011). The large contact surface area between the extraction solvent and aqueous sample forms a cloudy mixture. This phenomenon facilitates a rapid equilibration. When the cloudy solution is centrifuged, the extractant forms the sediment phase and removed with a microsyringe for later analysis (Yan and Wang 2013; Saraji and Boroujeni 2014).
The DLLME is a simple, fast, efficient, environmentally-friendly and economic method for sample preparation (Rezaee et al. 2006; Nagaraju and Huang 2007; Shamsipur and Fattahi 2011). It has been used for various types of biological matrices (Li et al. 2008; Xiong et al. 2009; Mashayekhi et al. 2010; Rezaee et al. 2010a; Rezaee et al. 2010b; Fernández et al. 2013). DLLME could be combined with a variety of chromatography techniques such as Gas chromatography- Mass Spectrometry (GC-MS) (Leong and Huang 2008; Meng et al. 2015), High Performance Liquid Chromatography (HPLC) (Ahmadi-Jouibari et al. 2013; Fernández et al. 2015) and capillary electrophoresis (Kohler et al. 2013).
Although, there are few studies about the analysis of opium alkaloids and opioids drugs in clinical biological samples (Wey and Thorman 2001; Whittington and Kharasch 2003; Saraji et al. 2011; Shamsipur and Fattahi 2011; Ranjbari et al. 2012; Ahmadi-Jouibari et al. 2013), but there are scant data about the analysis of opioids by DLLME-HPLC-PDA in postmortem urine samples. Therefore, in this study, we optimized a DLLME-HPLC-PAD for the extraction and determination of morphine, codeine, and methadone in postmortem urine samples.
Standard morphine, codeine and methadone were obtained from Darou Pakhsh Pharmaceutical Co. (Tehran, Iran). HPLC grade solvents including acetonitrile, methanol, acetone, chloroform, water, phosphoric acid, potassium dihydrogen phosphate, sodium carbonate were purchased from Merck Co. (Darmstadt, Germany). To prepare the 0.05 M phosphate buffer, 16.65 g potassium dihydrogen phosphate was dissolved in 2.5 l of HPLC-grade water and the pH of the buffer in the mobile phase was adjusted to pH 2.3 using phosphoric acid 85% w/v. Stock standard solution with concentration level 1 mg ml− 1 were prepared for morphine, codeine and methadone in methanol was prepared. Working standards were made by dilution of stock solution to final concentrations in urine. All solutions were stored at 4 °C.
An HPLC system including pump (Smartline, Model 1050) and Smartline PDA 2850 (multi wavelength) detector with RP column Eurospher® (250 mm × 4.6 mm, particle size: 5 μm) was used in this study. Data processing was performed with ChromGate® software (version 3.1.7), all from Knauer Co. (Berlin, Germany). The mobile phase consisted of acetonitrile (A) and 0.05 M phosphate buffer at pH 2.3 (B). Buffer and the mobile phase flow rate of 1 ml min− 1 was used in gradient elution mode: 0–7 min, A% 10 and B % 90; 7–8 min, A% 20 and B% 80; 8–15 min, A% 20 and B% 80; 15–16 min, A% 37 and B% 63; 16–40 min, A% 37 and B% 63; 40–45 min, A% 10 and B% 90.
Extraction of opioids in postmortem urine samples with DLLME
Blank postmortem urine samples (drug-free) were obtained during the autopsy of cadavers without any drug abuse/poisoning history. The blank samples tested by routine postmortem toxicological analysis (Thin layer chromatography (TLC) for screening and GC-MS for confirmation). Also, postmortem urine samples were collected from the cadavers with opioids abuse/poisoning which have been transferred to forensic toxicology laboratory of Zanjan legal medicine center (Zanjan, Iran). The samples were stored at − 20 °C until analysis. The ethical committee of the Legal Medicine Research Center (Tehran-Iran) approved this project (Grant No. 20726).
Initially, the frozen urine samples were thawed at room temperature and then were centrifuged for 15 min at 4000 rpm. The supernatant was transferred into clean 15 ml conical test tube and filtrated by a 0.22 μm filter, then 2 ml of the sample was transferred to a 10 ml test tube and 3 ml distilled water was added (to reduce matrix effects).
For the DLLME, an aliquot of 5 ml samples containing 2, 10 and 30 μg ml− 1 of morphine, codeine and methadone were prepared and pH of the samples was adjusted at 9.8 by adding appropriate amounts of sodium carbonate (10%w/v). 300 μl of chloroform (extraction solvent) and 500 μl of acetone (disperser solvent) were mixed well together then this mixture was injected rapidly by using a 2 ml syringe into the sample solution and a cloudy mixture has been formed. In this step, the analytes were extracted into the tiny droplet of chloroform, in a very short time. Then the samples were vortexed for 30 s and centrifuged at 4000 rpm for 10 min. After the centrifugation, fine droplets of extraction solvent were sediment at the bottom of the test tube. The sediment phase removed with a 100 μl microsyringe (Hamilton, USA) and replaced to a 1 ml glass vial. After evaporation of the solvent under stream of nitrogen gas, the residue was dissolved in 50 μl methanol and injected into the HPLC-PDA.
Optimization of DLLME
Affecting factors the DLLME procedure including the type and volume of extraction solvent, type and volume of disperser solvent, pH and extraction time were optimized in this study. Optimization of these factors was done by using postmortem blank urine samples spiked with morphine, codeine, and methadone.
Validation of DLLME- HPLC-PDA method
Limit of detection (LOD), limit of quantification (LOQ) and linearity
The limit of detection (LOD) and the limit of quantification (LOQ) were considered as the lowest concentration of the analytes corresponding to relationship of signal to noise ratio 3:1 and 10:1, respectively (SWGTOX 2013). The linearity of the method determined in the concentration ranges of 0.5–100 μg ml− 1 of morphine, codeine, and methadone. The calibration curves were drawn for morphine, codeine and methadone into blank postmortem urine samples spiked with concentrations of 0.5, 2, 5, 10, 20, 30, 40, 50 and 100 μg ml− 1 for each analyte. All concentrations were analyzed in triplicate.
Precision, accuracy, enrichment factor, recovery, and relative recovery
Inter-day and intra-day precisions method, and the enrichment factors and recovery for morphine, codeine and methadone were studied by extracting the spiked blank postmortem urine samples with 2, 10 and 30 μg ml− 1 concentrations. In order to evaluate the accuracy of the method acting to prepare three urine samples with concentrations of 2, 10 and 30 μg ml− 1 of morphine, codeine, and methadone (control urine samples). Then each of the three control samples was divided into three equal parts and was extracted by DLLME process. Accuracy in the format of the relative error (RE%) and precision to form of the relative standard deviation (RSD %) were reported.
The enrichment factor (EF) is the analytes concentration in the sediment and initial concentration of analytes within the sample and calculated according to previous study (Rezaee et al. 2006).
The extraction recovery (%ER) was defined as the ratio between the amount of the analyte in the sediment and the initial amount of the analyte within the sample and determined as previous method (Rezaee et al. 2006).
The relative recovery was studied by extracting the spiked postmortem urine samples (with suspected drug abuse) with two concentrations of morphine, codeine, and methadone (5 and 20 μg ml− 1). And then the relative recovery (%RR) was calculated according to the previous method (Rezaee et al. 2006).
Results and discussion
Selection of extraction solvent
Selection of a suitable extraction solvent is one of the key steps in the DLLME procedure that direct impact on the efficiency of method (Rezaee et al. 2006). The extraction solvent should be have a characteristics such as higher density and low solubility in water, miscible with disperser solvent and capability for extraction of target analytes (Saraji and Boroujeni 2014; Sharifi et al. 2016). Chloroform due to have characteristics such as the higher density than water, the boiling point and the solubility in water, was used as appropriate extraction solvent to extract opioids from the postmortem urine samples (Shamsipur and Fattahi 2011).
Selection of disperser solvent
The disperser solvent should be miscible in the water and dissolve in the extraction solvent and capable to form a cloudy solution (Saraji and Boroujeni 2014; Sharifi et al. 2016). In this study, acetone was selected as dispersive solvent due to the highest recovery of opium alkaloids, lower toxicity and cheaper than methanol and acetonitrile.
Optimization of extraction solvent volume
Optimization of disperser solvent volume
Performance of DLLME method directly influences by the volume of dispersive solvent. Changes in the volume of dispersive solvent cause changes the volume of the sediment phase. To obtain an optimal volume of acetone, was performed by several experiments using different volumes of acetone contains (300, 500, 750, 1000, 1500 and 2000 μl) and various volumes of chloroform (88, 100, 150, 200, 300 and 350 μl). All concentrations were analyzed in triplicate. The results showed that at low volume of acetone (300 μl) dispersion of the chloroform did not complete and a decrease in extraction recovery was observed. Also, in the high volume of acetone (1500 and 2000 μl), extraction recovery of morphine, codeine, and methadone decreased due to the increased solubility of the analyte in the sample solution and reducing entry them into the organic phase. Therefore, based on the results, 500 μl of acetone was selected as the optimal volume of disperser solvent in this study.
Optimization of pH
Optimization of extraction time
Validation of method
Quantitative results of morphine, codeine and methadone in spiked postmortem urine samples by DLLME- HPLC-PDA
LOQ (μg l− 1)
y = 22,063 x + 1135.8
y = 669,350 x - 217,207
y = 892,444 x - 50,709
Validation parameters of analytes in postmortem urine samples
Added concentration (μgml− 1)
Intraday precision RSD (%)
(n = 3)
Interday precision RSD (%)
(n = 3)
Accuracy (Relative Error %)
(SD, n = 3)
(SD, n = 3)
Relative recoveries and standard deviations of opioids in actual postmortem urine samples
Concentration of analyte (μg ml− 1)
Added concentration (μg ml−1)
Founded concentration (μg ml− 1)
N = 3 (SD)
Application of DLLME-HPLC-PDA procedure
After the optimization of the effective factors on DLLME and achieving to good and satisfactory results from the validated method, the DLLME-HPLC-PDA was used successfully for extraction and determination of morphine, codeine, methadone in 50 actual postmortem urine samples. Based on the obtained results, morphine was found in 22 samples, codeine was detected in 17 samples and methadone was detected in 27 samples. Some of the opium alkaloids such as papaverine, thebaine and noscapine were identified at 2, 1 and 3 samples, respectively. Also, 6-monoacetylmorphine and tramadol were determined in 8 and 6 samples, respectively. Concentration of morphine, codeine and methadone in postmortem urine samples were calculated in the range of: 0.28-26 μg ml− 1 (mean: 6.7 μg ml− 1) for morphine, 0.9–25.4 μg ml− 1 (mean: 13.52 μg ml− 1) for codeine and 0.4–43.8 μg ml− 1(mean: 33.5 μg ml− 1) for methadone.
Comparison of DLLME-HPLC-PDA method with other methods
Comparison of DLLME-HPLC-PDA with other analytical methods for determination of morphine, codeine and methadone in biological samples
LOD (μg l−1)
Dams et al. 2002
Fernandez et al. 2006
DLLME - SFO –HPLC-UV
Leong and Huang 2008
Shamsipur and Fattahi 2011
Ranjbari et al. 2012
Kohler et al. 2013
DLLME – HPLC-PDA
Fernández et al. 2013
Meng et al. 2015
DLLME – HPLC-PDA
In this study, the efficiency and performance of DLLME process were assessed under optimum conditions for the extraction of opioids from postmortem urine samples. According to repeatability, linearity, high extraction efficiency and good enrichment factor, this method is suitable for qualitative and quantitative analysis of opioids in postmortem urine samples. This is the first DLLME-HPLC-PDA method which optimized for postmortem urine samples and should be considered as an applied analytical method for determination of opioids in forensic toxicology laboratory.
We would like to show our gratitude to the Legal Medicine Research Center, Legal Medicine Organization, Tehran- Iran for financial support of this study.
This research was supported by Legal Medicine Research Center, Legal Medicine Organization, Tehran- Iran (Grant No. 20726).
Availability of data and materials
EA attributed in method validation, analysis of samples, data gathering and analysis and writing of the draft of the manuscript. MS attributed in sample collection and data gathering. AS attributed in method validation and analysis of the samples. GR attributed method validation and data gathering. KS attributed in study design, supervision on all of the research’s steps, revision the draft and writing the final version. All authors approved the final version of the paper.
EA (MSc in Toxicology) works as a technical expert in Laboratory of Toxicology. MS (MD, Forensic Medicine specialist) is head of the Zanjan Legal Medicine Center, Zanjan, Iran and member of the Legal Medicine Research Center, Legal Medicine Organization, Tehran Iran.AS (MSc in Toxicology) and GR (BS) are work as technical experts in Forensic Toxicology Laboratory of Zanjan Legal Medicine Center, KS (PharmD, PhD in Toxicology) is Associate Professor of Toxicology, Department of Forensic Toxicology, Legal Medicine Research Center, Legal Medicine Organization, Tehran Iran.
Ethics approval and consent to participate
All procedures performed in study involving human participants were in accordance with the ethical standards of the Legal Medicine Research Center’s ethical committee (Grant No. 20726) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Consent for publication
The authors declare that they have no competing interest.
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- Ahmadi-Jouibari T, Fattahi N, Shamsipur M, Pirsaheb M (2013) Dispersive liquid–liquid microextraction followed by high-performance liquid chromatography–ultraviolet detection to determination of opium alkaloids in human plasma. J Pharm Biomed Anal 85:14–20View ArticlePubMedGoogle Scholar
- Cheng J, Matsadiq G, Liu L, Zhou YW, Chen G (2011) Development of a model ultrasound-assisted surfactant-enhanced emulsification microextraction method and its application to the analysis of eleven polycyclic aromatic hydrocarbons at trace levels in water. J Chromatography A 1218:2476–2482View ArticleGoogle Scholar
- Dams R, Benijts T, Lambert WE, De Leenheer AP (2002) Simultaneous determination of in total 17 opium alkaloids and opioids in blood and urine by fast liquid chromatography–diode-array detection–fluorescence detection, after solid-phase extraction. J Chromatography B 773:53–61View ArticleGoogle Scholar
- Drummer OH (2004) Postmortem toxicology of drugs of abuse. Forensic Sci Int 42:101–113View ArticleGoogle Scholar
- Fernandez P, Morales L, Vazquez C, Bermejo AM, Tabernero MJ (2006) HPLC-DAD determination of opioids, cocaine and their metabolites in plasma. Forensic Sci Int 161:31–35View ArticlePubMedGoogle Scholar
- Fernández P, González C, Pena MT, Carro AM, Lorenzo RA (2013) A rapid ultrasound-assisted dispersive liquid-liquid microextraction followed by ultra-performance liquid chromatography for the simultaneous determination of seven benzodiazepines in human plasma samples. Anal Chim Acta 767:88–96View ArticlePubMedGoogle Scholar
- Fernández P, Regenjo M, Bermejo A, Fernández A, Lorenzo R, Carro A (2015) Analysis of drugs of abuse in human plasma by dispersive liquid–liquid microextraction and high performance liquid chromatography. J Applied Toxicol 35(4):418–425View ArticleGoogle Scholar
- Gergov M, Nokua P, Vuori E, Ojanpera I (2009) Simultaneous screening and quantification of 25 opioid drugs in post-mortem blood and urine by liquid chromatography–tandem mass spectrometry. Forensic Sci Int 186:36–43View ArticlePubMedGoogle Scholar
- He Y, Lee HK (1997) Liquid-phase microextraction in a single drop of organic solvent by using a conventional microsyringe. Anal Chem 69:4634–4640View ArticleGoogle Scholar
- Jeannot MA, Cantwell FF (1996) Solvent microextraction into a single drop. Anal Chem 68:2236–2240View ArticlePubMedGoogle Scholar
- Kohler I, Schappler J, Sierro T, Rudaz S (2013) Dispersive liquid-liquid microextraction combined with capillary electrophoresis and time-of-flight mass spectrometry for urine analysis. J Pharm Biomed Anal 73:82–89View ArticlePubMedGoogle Scholar
- Lee J, Lee HK, Rasmussen KE, Pedersen-Bjergaard S (2008) Environmental and bioanalytical applications of hollow fiber membrane liquid-phase microextraction: a review. Anal Chim Acta 624:253–268View ArticlePubMedGoogle Scholar
- Leong M, Huang SD (2008) Dispersive liquid-liquid microextraction method based on solidification of floating organic drop combined with gas chromatography with electron-capture or mass spectrometry detection. J Chromatography A 1211:8–12View ArticleGoogle Scholar
- Li Y, Hu J, Liu X, Fu L, Zhang X, Wang X (2008) Dispersive liquid-liquid microextraction followed by reversed phase HPLC for the determination of decabrominated diphenyl ether in natural water. J Sep Sci 31:2371–2376View ArticlePubMedGoogle Scholar
- Ma JJ, Du X, Zhang JW, Wang LZ (2009) Ultrasound-assisted emulsification microextraction combined with flame atomic absorption spectrometry for determination of trace cadmium in water samples. Talanta 80:980–984View ArticlePubMedGoogle Scholar
- Mabuchi M, Takatsuka S, Matsuoka M, Tagawa K (2004) Determination of morphine, morphine-3-glucuronide and morphine-6-glucuronide in monkey and dog plasma by high-performance liquid chromatography–electrospray ioniza- tion tandem mass spectrometry. J Pharm Biomed Anal 35:563–573View ArticlePubMedGoogle Scholar
- Mashayekhi HA, Abroomand-Azar P, Saber-Tehrani M, Hussain SW (2010) Rapid determination of carbamazepine in human urine, plasma samples and water using DLLME followed by RP-LC. Chromatographia 71:517–521View ArticleGoogle Scholar
- Meng L, Zhang W, Meng P, Zhu B, Zheng K (2015) Comparison of hollow fiber liquid -phase microextraction andultrasound-assisted low-density solvent dispersive liquid–liquid microextraction for the determination of drugs of abuse in biological samples by gas chromatography–mass spectrometry. J Chromatography B 989:46–53View ArticleGoogle Scholar
- Nagaraju D, Huang SD (2007) Determination of triazine herbicides in aqueous samples by dispersive liquid-liquid microextraction with gas chromatography-ion trap mass spectrometry. J Chromatography A 1161:89–97View ArticleGoogle Scholar
- Pathan H, Williams J (2012) Basic opioid pharmacology: an update. Br J Pain 6(1):11–16View ArticlePubMedPubMed CentralGoogle Scholar
- Ranjbari E, Golbabanezhad-Azizi AA, Hadjmohammadi MR (2012) Preconcentration of trace amounts of methadone in human urine, plasma, saliva and sweat samples using dispersive liquid–liquid microextraction followed by high performance liquid chromatography. Talanta 94:116–122View ArticlePubMedGoogle Scholar
- Rezaee M, Assadi Y, Hosseini MRM, Aghaee E, Ahmadia F, Berijani S (2006) Determination of organic compounds in water using dispersive liquid–liquid microextraction. J Chromatography A 1116:1–9View ArticleGoogle Scholar
- Rezaee M, Yamini Y, Faraji M (2010a) Evolution of dispersive liquid–liquid microextraction method. J Chromatography A 1217:2342–2357View ArticleGoogle Scholar
- Rezaee M, Yamini Y, Hojjati M, Faraju M (2010b) Novel extraction method based on the dispersión of the extraction solvent for extraction of letrozole from biological fluids. Anal Methods 2:1341–1345View ArticleGoogle Scholar
- Saraji M, Boroujeni MK (2014) Recent developments in dispersive liquid–liquid microextraction. Anal Bioanal Chem 406(8):2027–2066View ArticlePubMedGoogle Scholar
- Saraji M, Khalili Boroujeni M, Hajialiakbari Bidgoli AA (2011) Comparison of dispersive liquid–liquid microextraction and hollow fiber liquid–liquid–liquid microextraction for the determination of fentanyl, alfentanil, and sufentanil in water and biological fluids by high-performance liquid chromatography. Anal Bioanal Chem 400:2149–2158View ArticlePubMedGoogle Scholar
- Shamsipur M, Fattahi N (2011) Extraction and determination of opium alkaloids in urine samples using dispersive liquid–liquid microextraction followed by high-performance liquid chromatography. J Chromatography B 879(28):2978–2983View ArticleGoogle Scholar
- Sharifi V, Abbasi A, Nosrati A (2016) Application of hallow fiber liquid phase microextraction and dispersive liquid-liquid microextraction techniques in analytical toxicology. J Food Drug Anal 24:264–276View ArticlePubMedGoogle Scholar
- Shen G, Lee HK (2002) Hollow fiber-protected liquid-phase microextraction of triazine herbicides. Anal Chem 74:648–654View ArticlePubMedGoogle Scholar
- SWGTOX (2013) Scientific working Group for Forensic Toxicology (SWGTOX) standard practices for method validation in forensic toxicology. 2013. Available from: https://academic.oup.com/jat/articlepdf/37/7/452/2299550/bkt054.pdf, 11 Oct 2017
- UNODC (2016) World drug report 2016. United Nations publications, New YorkView ArticleGoogle Scholar
- Wey AB, Thorman W (2001) Capillary electrophoresis–electrospray ionization ion trap mass spectrometry for analysis and confirmation testing of morphine and related compounds in urine. J Chromatography A 916:225–238View ArticleGoogle Scholar
- Whittington D, Kharasch ED (2003) Determination of morphine and morphine glucuronides in human plasma by 96-well plate solid-phase extraction and liquid chromatography–electrospray ionization mass spectrometry. J Chromatography B 796:95–103View ArticleGoogle Scholar
- Xiong C, Ruan J, Cai Y, Tang Y (2009) Extraction and determination of some psychotropic drugs in urine samples using dispersive liquid– liquid microextraction followed by high-performance liquid chromatography. Jl Pharm Biomed Anal 49:572–578View ArticleGoogle Scholar
- Yan H, Wang H (2013) Recent development and applications of dispersive liquid–liquid microextraction. J Chromatography A 1295:1–15View ArticleGoogle Scholar
- Zhang Y, Lee HK (2012) Application of ultrasound-assisted emulsification microextraction based on applying low-density organic solvent for the determination of organochlorine pesticides in water samples. J Chromatography A 1252:67–73View ArticleGoogle Scholar