Accepted Manuscript

Title: Effects of Triglycerides Levels in Human Whole Blood

on the Extraction of 19 Commonly Used Drugs using
Liquid-liquid Extraction and Gas Chromatography-Mass
Spectrometry

Author: ZhiBin Huang Tianfang Yu Lin Guo Zebin Lin ZiQin

Zhao Yiwen Shen Yan Jiang Yonghong Ye Yulan Rao

PII: S2214-7500(15)00025-6
DOI: http://dx.doi.org/doi:10.1016/j.toxrep.2015.02.006
Reference: TOXREP 183

To appear in:

Received date: 15-12-2014

Revised date: 31-1-2015
Accepted date: 1-2-2015

Please cite this article as: Z.B. Huang, T. Yu, L. Guo, Z. Lin, Z.Q. Zhao,
Y. Shen, Y. Jiang, Y. Ye, Y. Rao, Effects of Triglycerides Levels in Human
Whole Blood on the Extraction of 19 Commonly Used Drugs using Liquid-
liquid Extraction and Gas Chromatography-Mass Spectrometry, Toxicol. Rep. (2015),
http://dx.doi.org/10.1016/j.toxrep.2015.02.006

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Effects of Triglycerides Levels in Human Whole Blood on the Extraction of 19
Commonly Used Drugs using Liquid-liquid Extraction and Gas
Chromatography-Mass Spectrometry

ZhiBin Huanga, Tianfang Yub, Lin Guoc, Zebin Lina, ZiQin Zhaoa*, Yiwen Shena*,
Yan Jianga, Yonghong Yea, Yulan Raoa*

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a
Department of Forensic Medicine, School of Basic Medical Sciences, Fudan

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University, Shanghai 200032, China
b

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Department of Clinical Medicine, Shanghai Medical College, Fudan University,
Shanghai 200032, China
c
Laboratory of Clinical Pharmacokinetics, Shuguang Hospital, Shanghai University
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of Traditional Chinese Medicine, Shanghai 201203, China
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ZhiBin Huang and Tianfang Yu contributed equally to this work.
Corresponding authors：Tel.: +86 21 54237403, fax: +86 21 54237404, E-mail address:
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yulan_rao@fudan.edu.cn (Y. Rao); Tel.: +86 21 54237668, fax: +86 21 54237668;

e

E-mail address: zqzhao@shmu.edu.cn (Z. Zhao); Tel.: +86 21 54237402, fax: +86 21
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54237404, E-mail address: shenyiwen@fudan.edu.cn (Y. Shen).

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Abstract
Liquid-liquid extraction (LLE) is the most commonly sample preparation
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procedure used by forensic toxicologists in China for screening drugs in whole human
blood. It extracts numerous substances from blood including targeted drugs and
interfering substances, specifically triglycerides (TG). With increasing prevalence of
hyperlipidemia, the influences of TG on LLE and on subsequent analysis with gas
chromatography-mass spectrometry (GC-MS) may become a major issue for forensic
laboratories. This study aims to elucidate the influences of TG on LLE and to provide
possible solutions to this problem. Nineteen commonly encountered drugs in forensic
cases were spiked to human whole blood with different TG concentrations. Diethyl
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Page 1 of 20
ether, ethyl acetate/hexane mixed solutions, chlorobutane and several other frequently
used solvents were tested for the extraction of drugs from spiked whole blood. The
supernatant organic layer was evaporated to dryness and reconstituted with methanol.
The resultant products were analyzed by GC-MS, and the extraction recovery was
calculated. LLE with diethyl ether, ethyl acetate/hexane (9:1) and chlorobutane all
possessed effective and reliable extraction recoveries for blood sample with low TG

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concentrations (0.63-6.85mmol/L). At high TG concentrations, diethyl ether produced
a highly turbid substance that could not be further analyzed using GC-MS. Extraction

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recoveries drastically dropped for ethyl acetate/hexane (9:1) mixture at high TG
concentrations, while chlorobutane experienced minimal drops in extraction

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recoveries. In conclusion, TG levels in whole blood noticeably influence drug
recovery to variable extents depending on the LLE solvent. Chlorobutane showed
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minimal influences from TG content in whole blood and thus is the recommended
LLE solvent for forensic drug extraction.
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Keywords: Triglycerides, Liquid-liquid extraction, GC-MS, Forensic toxicology
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1. Introduction
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Human whole blood is a ubiquitous sample in the field of forensic toxicology.

The most common procedure of choice for pretreating whole blood is initial
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pretreatment by liquid-liquid extraction (LLE). Because of its suitability for screening,

ease of operation, low cost, and adaptability (Saar et al. 2009), LLE is accredited as
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the standard technique to pretreat whole blood from forensic cases for drug and
poison detection in China.
In LLE, an extraction solvent is used to extract and purify the analytes out of
whole blood to be further analyzed. One main parameter for assessing LLE efficacy is
the extraction recovery of targeted analytes. Other parameters that need to be
considered include the extraction solvent’s specificity, volatility and toxicity
(Couchman and Morgan 2011). Unfortunately, extraction solvents often can extract
additional endogenous substances, such as triglyceride (TG) (Megremis 1991), which
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Page 2 of 20
may interfere with subsequent analysis (Ali and Cole 2001). With increasing
prevalence of hypertriglyceridemia in China (up to 11.3% for individuals over 18
years old) (Li et al. 2012a; Li et al. 2012b), the level of TG is now more likely to
affect the detection of drugs in whole blood. It was believed the saturated fatty acid
chains of TG showed affinity to form tight bonds with certain drugs, depending on
chemical structure and polarity of the drug. This could result in formation of

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complexes that cannot be extracted efficiently by LLE solvents (Donald 2003; Foye
2002). This study hypothesizes that high TG levels can reduce the extraction

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recoveries of drugs when using LLE.
The primary purpose of this study was to determine the impact of TG content on

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LLE for human whole blood. This study also attempts to find an alternative extraction
solvent that can counteract the negative impacts of TG.
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2. Materials and Methods
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2.1 Chemicals and solutions
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Amphetamine (AMP), methamphetamine (MAMP),

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3,4-methylendioxyamphetamin (MDA), 3,4-methylenedioxymethamphetamine

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(MDMA), ketamine, methadone, pethidine, secobarbital, lidocaine, clenbuterol,

benzhexol, carbamazepine, diazepam, chlorpromazine, olanzapine, flurazepam,
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clozapine, alprazolam, triazolam and diphenoxylate were purchased from the National
Institute for Control of Pharmaceutical and Biological Products (Beijing, PR China).
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HPLC grade chlorobutane was obtained from Sigma-Aldrich Co., Ltd. (St. Louis,
USA). Analytical grade ethyl acetate, hexane, cyclohexane, heptane, isooctane,
sodium hydroxide (NaOH) and HPLC grade methanol were purchased from
Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

2.2 Instrumentation and chromatographic conditions

The chromatographic system used was an Agilent 7890 GC. It was fitted with a
5975C mass detector (MSD) (Agilent Technologies, Palo Alto, CA, USA) and
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Page 3 of 20
connected to HP Chemstation software for data recording.
Separations were conducted on a HP-5MS capillary column (30m×0.25mm×
0.25μm) (Agilent Technologies, Palo Alto, CA, USA). 1 μL of sample was injected in
split mode (split ratio=10:1) using an ionizing energy of 70 eV with temperatures of
the inlet, MSD transfer line, quadrupole and ion source at 250°C , 280°C, 150°C and
230°C, respectively. Temperature of the column was set at 100°C initially, maintained

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for 1 min and increased at a rate of 20°C/min to 280°C, which was kept constant for
23 minutes. Helium was used as the carrier gas at a constant flow rate of 1 mL/min for

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a total GC runtime of 33 min. There was a 3 min solvent delay before the ion source
was turned on. Selected ion monitoring (SIM) mode was utilized to collect

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chromatograms. Two or three fragment ions were used for each compound (Table 1).

2.3 Specimen
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Whole blood samples for preliminary experiments were leftover blank blood
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from forensic cases. Hypertriglyceridemia whole blood samples were obtained from
96 volunteers, and they were divided into 5 groups according to TG concentrations,
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which were measured with a Hitachi 7600-120 Model Automatic Analyzer. Overall
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TG concentration ranged from 0.60mmol/L to 33.35mmol/L. All samples were stored

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at -20°C for two months prior to LLE.

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2.4 Sample preparation

2mL of human whole blood was initially spiked with 19 drugs each reaching a
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concentration of 5µg/ml except diphenoxylate which reached 7.5µg/ml. 3mL of

extraction solvent was then added to the sample. The samples were mixed for 2
minutes followed by centrifugation at 4,000 RPM for 5 minutes. The supernatant
organic layer was collected. 200µl of NaOH (10%) and 3mL of the same extraction
solvent used previously was added to the pellet remaining from centrifugation. The
samples were again mixed for 2 minutes followed by centrifugation at 4,000 RPM for
5 minutes. The supernatant organic layer was collected and combined with the
previously collected supernatant. The combined supernatant was then evaporated to
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Page 4 of 20
dryness under air at 50°C. The residue was reconstituted in 100μL of methanol, of
which 1μL was injected into the GC-MS system. The extraction recovery values were
calculated by comparing the peak areas of the analytes extracted from whole blood to
the areas obtained by injecting the standard solutions at the same concentrations.

2.5 Statistical Analysis

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Statistical analyses in this study were conducted using Statistical Product and
Service Solutions (SPSS) version 15.0.

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3. Results & Discussion

3.1 Preliminary selection of organic solvents

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The organic solvents used for the extraction of drugs in human whole blood
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were selected based on frequency of usage in research and blood detection in forensic
practice (Alves et al. 2012; Chen et al. 2000; Chlobowska et al. 2004; Malakova et al.
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2007; Owens et al. 2007; Papoutsis et al. 2012; Sporkert et al. 2012; Tennakoon et al.
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2013; Versace et al. 2012; Watzer et al. 2002; Zhang and Lee 2012) , and on their
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corresponding toxicities. Thus, solvents of high toxicity such as benzene, chloroform

and dichloromethane were not included in our list despite favorable extraction
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efficiency. Our preliminary list contained pure solvents (diethyl ether, chlorobutane,
hexane, ethyl acetate, heptane, and isooctane), and mixed solutions
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(chlorobutane/isopropanol solution and ethyl acetate/hexane solution). These

extraction solvents were evaluated based on recoveries using the same spiked whole
blood.

3.1.1 Evaluation of pure solvent

Six pure solvents were studied, and it was found that, the extraction solvents
that possessed the highest and most consistent recoveries were chlorobutane, diethyl
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Page 5 of 20
ether and ethyl acetate (Table 2). Hexane possessed low recovery specifically for
chlorpromazine, flurazepam, and carbamazepine, while heptane and isooctane
possessed low recovery for alprozolam and chlorpromazine. Although heptane,
hexane and isooctane are commonly used as the extraction solvents in other
researches, we believe their weak polarities constitute to their low recoveries, making
them unsuitable for LLE. Thus, chlorobutane, ethyl acetate and diethyl ether were

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selected as the extraction solvents for further testing.

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3.1.2 Evaluation of ethyl acetate/hexane mixed solutions

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For ethyl acetate/hexane solution, the ratio of ethyl acetate to hexane varied
greatly in research (Owens et al. 2007; Watzer et al. 2002) and required a selection of
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an optimal mixture. The recoveries for pure ethyl acetate were compared to those of
ethyl acetate/hexane mixture at ratios of 2:1, 6:1 and 9:1. The results showed little
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difference between pure ethyl acetate and three ethyl acetate/hexane mixtures,
showing a relative standard deviation (RSD) lower than 11.1%. Using MDMA as an
example, the recovery was 51.5% when using ethyl acetate alone, and 51.7%, 49.2%
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and 51.3% at ratios of 2:1, 6:1, 9:1, respectively (RSD=2.2%, n=4). The only notable
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improvement was the 70% increase in extraction recovery for chlorpromazine by

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ethyl acetate/hexane mixture at 9:1.

Furthermore, from the gas chromatogram, we discovered that the ethyl

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acetate/hexane mixtures consistently produced significantly lower background noise

than pure ethyl acetate from impurities (Fig 1). This phenomenon was most
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recognizable for column bleeding at retention time 5.2 min. We believed that the
addition of hexane to ethyl acetate helped protect the gas chromatographic column but
further investigation would be required for confirmation.
Therefore, ethyl acetate/hexane mixed solution (9:1) replaced pure ethyl acetate
in further testing.

3.1.3 Evaluation of chlorobutane/isopropanol (4:1) mixed solution

Page 6 of 20
 We discovered that the addition of chlorobutane to blood followed by mixing
often resulted in emulsification of the sample. This phenomenon formed a thick,
turbid layer that effectively trapped and prevented the drugs from separating into the
supernatant organic layer during centrifugation. As a result, the drugs remained in the
pellet and recoveries drop drastically. To deal with this problem,
chlorobutane/isopropanol (4:1) mixture was used instead of pure chlorobutane

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(Versace et al. 2012). Isopropanol acted as a de-emulsifier and subsequent mixing
effectively counteracted the emulsification. However, this mixed solution produced

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varying results for drug recoveries. The mixed solution reduced recoveries for
MDMA, clenbuterol, carbamazepine, clozapine and alprazolam while only increasing

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recoveries for MDA and diphenidol. Furthermore, the mixed solution produced more
background noise on chromatogram than pure chlorobutane. Therefore, pure
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chlorobutane was preferable for further testing. In regards to emulsification, saturated
salt water equal to the amount of chlorobutane was added to the emulsified substance.
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Subsequent mixing and centrifugation counteracted emulsification while also
preserving recoveries.
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3.2 Effect of tg concentration on the extraction recovery when using diethyl ether,
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ethyl acetate/hexane mixture and chlorobutane as the extraction solvents

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19 drugs spiked to whole blood samples with varying TG concentrations were

extracted by the solvents obtained from preliminary selection, and the recoveries were
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compared

3.2.1 Diethyl ether as the extraction solvent

Under low TG concentrations (1.67, 3.79, and 6.87mmol/L), diethyl ether
produced recoveries ranging from 11.7% to 81.1%, and 16 out of 19 drugs had
consistent recoveries above 50%. The recovery data was highly precise. The RSDs of
the recoveries for MDA, MDMA, carbamazepine, chlorpromazine, clozapine and
diphenxylate were 7.6%, 11.3%, 11.4%, 14.1%, 16.2% and 16.2% respectively for the
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Page 7 of 20
three trials. However, diethyl ether produced low recoveries for olanzapine, AMP, and
MAMP at 32.7% (RSD=16.8%, n=3), 21.3% (RSD=35.0%, n=3) and 11.7%
(RSD=47.9%, n=3) respectively. Regarding the poor recovery associated with
amphetamines, we, as well as other researchers, have observed this phenomenon
where amphetamines are extremely volatile by nature and can easily evaporate along
with the supernatant during evaporation. (Holler et al. 2005; Wohlfarth et al. 2010;

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Guo et al. 2015).
In this study, diethyl ether consistently produced serious procedural problems

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when pretreating whole blood under high TG concentrations. This problem occurred
for all trials with TG concentrations of 11.43mmol/L, 15.06mmol/L, 19.76mmol/L

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and 26.78mmol/L. Evaporation of the supernatant organic layer to dryness after LLE
with diethyl ether resulted in a brownish yellow, turbid and oily substance that could
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not reconstitute in methanol. This resulted in a turbid mixture which could not be
injected for further instrumental analysis. In fact, this phenomenon has been
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frequently encountered in routine practice during forensic blood analysis in our lab. In
certain cases such as hemorrhagic shock and cardiac rupture, the collected blood
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sample is extremely scarce, sometimes less than 2 mL. According to the standard
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protocol, a 2 mL of sample can supply enough blood for only one analytical procedure.
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Thus, if the extracted residue becomes turbid after LLE with diethyl ether, the blood
analysis would become inconclusive as there may be inadequate blood left for
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additional analysis. Therefore, diethyl ether can only be used reliably for whole blood
sample with low TG concentrations.
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3.2.2 Ethyl acetate/n-hexane mixture (9:1) as the extraction solvent

At low TG concentrations ranging from 1.12 to 6.85mmol/L, ethyl
acetate/hexane (9:1) mixed solution consistently produced high recoveries, especially
for MAMP, clozapine, lidocaine, ketamine, carbamazepine and chlorpromazine,
which averaged to 90.5±4.7%, 91.5±3.6%, 91.7±2.7%, 94.5±3.0%, 92.7±5.7% and
91.5±4.6%, respectively. However, a rise in TG concentration resulted in dramatic
drops in recoveries (Fig 2). This was especially evident in the highest TG
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Page 8 of 20
concentration of 23.35mmol/L where recoveries dropped for all 19 drugs. This
showed that high TG concentrations could dramatically reduce the recoveries when
using ethyl acetate/hexane (9:1) mixed solution.

3.2.3 Chlorobutane as the extraction solvent

Chlorobutane produced the most consistent and reliable drug recoveries at all

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levels of TG concentration. Most drugs produced recoveries ranging from 30.3% to
85.8%, with the exception of four amphetamines (AMP, MAMP, MDA and MDMA)

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at any TG concentrations and of olanzapine at high TG concentrations (Table 3).
These reduced recoveries for the amphetamines were also found in a study by Demme

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where chlorobutane was used to extract over 200 drugs from water (Demme U 2005).
Demme discovered that chlorobutane was an extremely potent extraction solvent for
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most drugs except amphetamines. As previously mentioned for diethyl ether, this was
likely due to the loss of amphetamines during evaporation. The recoveries for most
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drugs only dropped minimally with increasing TG concentration. Most notable among
varying TG concentrations were for dolantin, ketamin, lidocaine, clenbuterol,
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diazepam, flurazepam and triazolam whose recovery RSDs were only 13.9%, 15.4,
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13.6%, 13.2%, 15.6%, 15.6% and 16.5%, respectively. However, the extraction
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recoveries of olanzapine dropped to 1.0% and 3.6% when TG concentration reached

15.06 and 33.35 mmol/L. The non-polar piperazine ring of olanzapine may strengthen
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the affinity of the drug to bind to the saturated fatty acid chains of TG. This resulted
in the formation of drug-TG complexes that could not be extracted by LLE. Thus,
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olanzapine showed drastically reduced recoveries under high TG concentrations.

Meanwhile, secobarbital, clenbuterol, diphenoxylate also possessed relatively low
recoveries, which averaged 45.6% (RSD=23.8%, n=8), 55.0% (RSD=13.2%, n=8),
and 30.3% (RSD=30.3%, n=8), respectively. The 2-pentyl group and the 3-propenyl
group of secobarbital, the tert-butyl group of clenbuterol and the tetrahydropyridine
structure of diphenoxylate increased each of their overall lipophilicity. Thus, we
believe these drugs were also more likely to bind to TG to form complexes that could
not be effectively extracted, resulting in decreased recoveries. These results suggested
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Page 9 of 20
the extraction efficacy of chlorobutane was thwarted by TG levels only towards
specific drugs such as olanzapine but remained consistent for most other drugs.

3.2.4 Cross comparison of diethyl ether, ethyl acetate/n-hexane and chlorobutane

For a particular blood sample, the recoveries of all 19 drugs were horizontally
compared for diethyl ether, chlorobutane and ethyl acetate/hexane (9:1) mixed

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solution by extracting chemically stable drugs, particularly diazepam and benzhexol
(Table 4). The recoveries of the drugs extracted by the three extraction solvents

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differed negligibly in samples with TG concentrations ranging from 3.79 mmol/L to
6.87 mmol/L. Diethyl ether showed lower recoveries for diazepam and benzhexol

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than the other two solvents in samples with TG concentration at 1.67 mmol/L and
7.75 mmol/L (Table 4). When TG concentration was above 6.87 mmol/L, recoveries
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obtained using diethyl ether as the extraction solvent declined drastically, which
suggested that diethyl ether was greatly affected by TG concentration. Chlorobutane,
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on the other hand, produced the most consistent recoveries for all drugs even at high
TG concentrations (Table 3), thus making it the extraction solvent of choice when
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facing unknown or high concentrations of TG in human whole blood.

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4. Conclusions
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Initial findings showed that ethyl ether, ethyl acetate and chlorobutane produced
higher recoveries than hexane, heptane and isooctane. Furthermore, the addition of
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hexane to ethyl acetate drastically reduced background noise in gas chromatogram

while maintaining recoveries. Therefore, diethyl ether, ethyl acetate/n-hexane (9:1)
and chlorobutane were compared with respect to their ability to extract drugs from
human whole blood under different TG concentrations. Extraction by diethyl ether
performed well for blood samples with low TG concentrations, but often formed
turbid residues when TG concentrations were high, which prevented further
instrumental analysis. Ethyl acetate/hexane (9:1) mixed solution also effectively
extracted all 19 drugs from blood samples with low TG concentrations but recoveries
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Page 10 of 20
drastically fell with increases in TG concentration. Finally, chlorobutane effectively
and reliably extracted most drugs, except the amphetamines, from blood samples with
TG content in the range of 0.63-26.78 mmol/L. The amphetamines typically
possessed low recovery and therefore traditional LLE might not be suitable for the
extraction of amphetamines from whole blood samples. Chlorobutane sporadically
formed an emulsified substance that drastically decreased recovery when mixed with

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blood. However, the addition of saturated salt water effectively eliminated the
emulsification and restored recovery. Despite this drawback of chlorobutane, it still

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produced reliable and consistent recovery for drugs spiked to whole blood at varying
TG concentrations. Therefore, we recommended chlorobutane as the primary

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extraction solvent for LLE pretreatment of human whole blood for forensic purposes.

Conflict of interest
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We declare that we have no conflict of interest.
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550000

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450000

400000

350000

300000

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200000

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100000

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9.0 9.2 9.4 9.6 9.8 10.0 10.2 10.4 10.6 10.8 11.0 11.2
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Fig1. Mass spectrum of pure ethyl acetate versus ethyl acetate/hexane 9:1

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Ac

Fig2. Recovery of 19 drugs at varying TG concentrations using ethyl acetate/n-hexane

14

Page 14 of 20
(9:1) mixed solution

Table 1 Retention time and fragment ions of the 19 drugs chosen to be spiked into
human whole blood

Spiked

t
Retention time

ip
Drug Significant ions (m/z) concentration
(RT)（min）
（μg/mL）

cr
AMP 4.30 44\91\65 5.0

us
MAMP 4.55 58\91\149 5.0
MDA 6.20 44\136\179 5.0
MDMA
Pethidine
6.45
7.60
an
58\135\193
71\247\172
5.0
5.0
Secobarbital 7.80 168\195\124 5.0
M
Ketamine 8.25 180\209\152 5.0
Lidocaine 8.30 86\58\234 5.0
d

Clenbuterol 9.20 86\57\127 5.0

e

Benzhexol 10.20 98\218\118 5.0

pt

Carbamazepine 10.60 193\236\165 5.0

Diazepam 11.20 256\283 5.0
ce

Chlorpromazine 11.50 58\86\318 5.0

Olanzapine 13.35 242\229\312 5.0
Ac

Flurazepam 13.65 86\99\387 5.0

Clozapine 14.9 243\256\192 5.0
Alprazolam 16.0 279\204\308 5.0
Triazolam 17.6 313\238\342 5.0
Diphenoxylate 27.8 246\42\91 7.5

AMP, amphetamine;

15

Page 15 of 20
 MAMP, methamphetamine;
MDA, 3,4-methylenedioxyamphetamine;
MDMA, 3,4-methylenedioxymethamphetamine.

t
ip
Table 2 Recoveries of most common drugs for comparison between extraction
solvents

cr
Diethyl Ethyl
Chlorobutane Heptane Hexane Isooctane

us
ether acetate
Diazepam 56.3% 46.9% 65.1% 38.8% 22.1% 14.3%
Chlorpromazine
Flurazepam
47.9%
59.2%
42.6%
47.5%
36.7%
52.6%
an 23.2%
38.4%
3.2%
4.4%
8.4%
15.8%
Clozapine 50.1% 45.5% 60.5% 24.5% 34.8% 1.3%
M
Alprazolam 33.9% 33.0% 54.6% 0.10% 56.4% 0.12%
Clenbuterol 42.5% 59.0% 69.8% 44.9% 12.3% 2.7%
d

Carbamazepine 34.5% 40.6% 78.8% 0.6% 0.10% 0.08%

e
pt
ce
Ac

16

Page 16 of 20
Table 3. Recovery of 19 drugs at varying TG concentrations using chlorobutane.

Drug\TG
RSD
concentratio 0.63 1.47 1.99 2.24 3.81 7.04 15.06 26.78 Mean
（%）
n (mmol/L)

AMP 32.2 18.4 35.4 35.8 33.4 31.9 6.5 29.4 26.4 38.8

MAMP 32.3 16.6 32.3 37.3 49.2 30.3 31.2 29.4 30.3 29.8

t
ip
MDA 3.6 8.6 18.7 43.7 29.8 46.0 0.2 49.9 23.0 87.2

MDMA 12.4 46.0 67.5 57.0 69.4 38.7 32.9 75.7 48.9 44.0

cr
Pethidine 63.4 71.1 82.6 76.0 86.4 95.6 79.5 65.7 78.0 13.9

us
Secobarbital 40.6 44.5 49.8 42.6 48.7 62.2 44.1 23.4 45.6 23.8

Ketamine 60.8 75.6 88.5 72.7 86.2 97.2 80.3 65.8 78.6 15.4

Lidocaine 69.6 85.7 95.5 80.9 92.7 102.3

an 83.6 70.0 85.8 13.6

Clenbuterol 46.6 57.0 65.2 53.5 62.1 53.2 44.0 58.8 55.0 13.2

Benzhexol 71.1 80.0 83.4 72.2 85.6 102.9 77.7 53.2 77.5 18.3
M
Carbamazepi
55.6 74.4 77.8 61.2 66.5 89.8 60.1 49.6 68.2 19.2
ne
d

Diazepam 73.2 87.6 96.8 78.9 89.3 99.8 73.6 62.2 82.6 15.6
e

Chlorpromaz
67.8 70.8 74.2 67.2 71.9 83.1 49.3 40.9 65.0 21.2
pt

ine

Olanzapine 38.7 63.3 79.4 44.3 23.4 80.7 1.0 3.6 45.0 69.6
ce

Flurazepam 71.3 83.9 92.6 78.2 85.8 100.5 70.0 62.1 81.4 15.6

Clozapine 69.5 92.8 109.4 75.3 82.2 117.2 54.9 57.4 83.8 27.2
Ac

Alprazolam 66.7 86.6 95.6 75.5 83.4 105.8 75.5 59.4 82.5 18.3

Triazolam 65.6 78.9 86.5 68.4 76.5 91.3 68.1 52.6 75.0 16.5

Diphenoxyla
25.9 30.3 39.7 28.6 31.7 74.4 36.2 15.2 30.3 24.7
te

TG, triglycerides; RSD, relative standard deviation; AMP, amphetamine; MAMP,

methamphetamine; MDA, 3,4-methylenedioxyamphetamine; MDMA,

17

Page 17 of 20
3,4-methylenedioxymethamphetamine.

Table 4. Recoveries of diazepam and benzhexol at four TG concentrations obtained

using diethyl ether, ethyl acetate/hexane mixed solution (9:1) and chlorobutane as the
extraction solvents.

t
ip
Diazepam Benzhexol

cr
TG concentration
1.67 3.79 6.87 7.75 1.67 3.79 6.87 7.75
(mmol/L)

us
57.7 69.6 82.0 58.4 56.8 68.4 82.0 58.2
Diethyl ether
% % % % % % % %
Ethyl acetate/hexane
mixture (9:1)
76.0
%
73.1
%
76.6
%
an
87.6
%
80.1
%
75.6
%
79.2
%
90.9
%
M
81.3 76.4 71.4 98.6 82.4 73.3 73.2 84.0
Chlorobutane
% % % % % % % %
e d
pt
ce
Ac

18

Page 18 of 20
Fig1

i
cr
us
an
M
ed
pt
ce
Ac

Page 19 of 20
Fig2

i
cr
us
an
M
ed
pt
ce
Ac

Page 20 of 20