Wang.

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Journal of Analytical Toxicology, Vol. 35, January/February 2011

The Quantitative Analysis of Paraquat in

Biological Samples by Liquid Chromatography–
Electrospray Ionization-Mass Spectrometry

Zhaohong Wang1,*, Zhiping Wang2, and Junbo Xing3

1Procuratoral Technology and Information Research Center, Supreme People’s Procuratorate, Lugudonglu 5, Shijingshan District,

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Beijing 100040, China; 2Toxicology Division, Forensic Science Institute of Public Security Ministry, Muxidi Nanli 17,
Xicheng District, Beijing 100038, China; and 3Institute for Drug and Instrument Control, Health Department of GLD, PLA,
Beijing 100071, China

Abstract Treatment of PQ poisoning remains ineffective, and current

approaches rely on diminishing the absorption of the re-
A quantitative method for the analysis of paraquat in blood and maining PQ in the gastrointestinal tract, removal of blood PQ
tissue samples has been developed using liquid chromatography– by hemoperfusion and forced diuresis, and maintenance of
electrospray ionization-mass spectrometry. Chromatographic vital functions. The fatal outcome, however, is still dose-de-
separation was performed on an Atlantis HILIC silica column using pendent, and several indexes correlating plasma PQ concen-
a mobile phase of acetonitrile/250 mM ammonium formate in the trations with the probability of death exist (4). Therefore, iden-
gradient mode. The method was linear from 2 to 500 ng/mL, with a
tification and quantification of PQ in biological samples is
correlation coefficient of 0.998. The limits of detection and
quantitation in blood were determined to be 0.5 and 2 ng/mL,
necessary for forensic toxicology and therapeutic drug moni-
respectively. Intraday precision was performed in one extraction by toring.
analyzing five aliquots of three controls with different A number of analytical methods have been previously re-
concentrations in the established linear range. Interday precision ported in the literature for identification and quantitation of PQ
was determined by analyzing one aliquot of each control over 10 in biological specimens. Methodology includes radioim-
extractions. The coefficients of variation were less than 10% for munoassay (5), spectrophotometry (6,7), gas chromatography
both intra- and interday assays. The method has been successfully (GC) (8), high-performance liquid chromatography (HPLC)
applied to blood and tissues samples from nine cases and can be (2,9–16), capillary electrophoresis (17), and liquid chro-
used to detect the presence of paraquat in forensic testing. matography–mass spectrometry (LC–MS) (18,19).
This paper presents a new LC–electrospray ionization-mass
spectrometry (ESI-MS) method and demonstrates its utility by
presenting the postmortem tissue distribution of PQ in five
Introduction
cases.
Paraquat (PQ) is a bis-quaternary ammonium compound
that has been in widespread use since 1962 as a domestic and
commercial herbicide. Occasionally, people are exposed to it, Experimental
and there have been many reports about accidental and suicidal
ingestions (1–3). It is believed that an oral dose of only 1–2 g Reagents and materials
is fatal to most adults (1). Victims of poisoning experience epi- Paraquat dichloride and ethyl viologen dibromide, the in-
gastric pain, vomiting, dyspnea, dysuria, and jaundice; death ternal standard, (Figure 1) were obtained from Sigma (St.
usually follows, often after a period of many days, as a result of Louis, MO). All solvents were HPLC grade and purchased from
severe and extensive fibrotic lung damages and renal failure. Fisher Scientific (Pittsburgh, PA). Ammonium formate was
Some subjects become comatose and die within a few hours, purchased from Fluka (Buchs, Switzerland). Oasis® WCX solid-
prior to the development of significant organ pathology (4). phase extraction (SPE) columns were purchased from Waters
(Milford, MA). Deionized water was prepared through a Milli-
* Author to whom correspondence should be addressed. Email: [email protected]. pore water purification system.

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Journal of Analytical Toxicology, Vol. 35, January/February 2011

Specimens tonitrile aqueous solution, and the eluates were evaporated

During 2007, nine cases of suspected paraquat intoxication under nitrogen at 45°C. The residues were reconstituted with
were referred to our laboratory from public security bureau 50 μL of 1:1 ratio of mobile phase (acetonitrile and 250 mM
throughout China. Five of the nine were reported as positive ammonium formate aqueous solution).
for the presence of paraquat. Drug-free blood specimens were
sampled from 10 volunteers who had not come into contact Instrumental analysis
with any drugs. These blood samples were confirmed to be The samples were analyzed for PQ using a Waters UPLC
drug-free by GC–MS and LC–MS–MS before use. system coupled with an ACQUITY BSM_SM_Quattro Premier
XE MS (Milford, MA). The LC system is comprised of a binary
Standards, calibrators, and control preparation solvent manager, sample manager, and column manager. Five-
A stock standard of PQ was prepared in deionized water and microliter injections were made onto an ACQUITY BEH HILIC
stored in polypropylene vials in the dark at –80°C. Deuterated column (Waters, 50 mm × 2.1-mm i.d., dp = 1.7 μm) that was
PQ is not commercially available; therefore, a different com- held at 30°C. The mobile phase consisted of 250 mM ammo-
pound was chosen as the internal standard. Several factors nium formate aqueous solution (adjust to pH 3.7 with formic
were used to make this decision, including structural similar- acid) and acetonitrile. A mobile phase gradient was used with
ities, extraction performance, and availability to the laboratory. a flow rate of 0.30 mL/min. Immediately after injection, the

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Ethyl viologen dibromide was chosen as the internal standard acetonitrile percentage was decreased from an initial value of
because it shares chemical structure properties (Figure 1) and 60% to 20% over 0.5 min, then increased to 60% in 1.5 min
behaves similarly to PQ during extraction. More importantly, with a post-equilibration time of 1.5 min. The total run time
its presence is highly unlikely in the case samples because it is was 3 min.
not used commercially. The compound was also immediately An electrospray interface in the positive mode was used with
available to the laboratory. A stock standard of ethyl viologen a cone gas at 50 L/h and a desolvation gas at 651 L/h at 350°C.
dibromide was prepared in deionized water and stored in Optimization of MS–MS conditions were performed by a flow
polypropylene vials in the dark at –80°C. Calibrator and control injection of PQ and ethyl viologen in acetonitrile/250mM am-
samples were prepared in 1.0 mL of certified drug-free blood by monium formate aqueous solution (40:60, v/v) into the elec-
spiking PQ at concentrations of 2, 10, 50, 100, and 500 ng/mL. trospray source.
Three controls were prepared at 5, 50, and 400 ng/mL and The general MS–MS conditions were as follows: the capillary
were used throughout the method validation procedure. The voltage was 3.5 kV and the cone voltage 20 V. The extractor
calibrators and controls were spiked prior to each extraction. voltage was set at 4.0 V and the RzF lens voltage 0.5 V. The
analysis was performed by multi-reaction monitoring, using
Sample preparation and extraction the precursor ion at m/z 186 and the product ion at m/z 155
Blood, urine, and tissue homogenates (1 mL or 1 g) were and 171 for PQ and the precursor ion at m/z 214 and product
prepared by the addition of 3 mL of 0.1 M phosphate buffer (pH ions at m/z 158 and 185 for the internal standard, ethyl vio-
7), and 100 ng/mL of the internal standard solution; thereafter, logen. The transitions for quantitation were 186 → 171 for PQ
all samples were centrifuged at 8000 rpm for 10 min. Three- and 214 → 158 for ethyl viologen. Table I outlines the MRM pa-
milliliter Waters Oasis WCX columns were sequentially condi- rameters for PQ and the internal standard.
tioned with 3 mL methanol, 3 mL deionized water, and 1 mL
of 0.1 M phosphate buffer (pH 7). Following the sample appli- Method validation
cation, the columns were washed with 3 mL of 0.1 M phosphate The method was validated using certified drug-free whole
buffer (pH 7), 3 mL deionized water, and 3 mL of methanol. blood. The calibrators and controls were spiked prior to each
The columns were dried under vacuum for 10 min. The ana- extraction. The method validation included the determination
lytes were eluted with 2 mL of 2% formic acid in an 80% ace- of the limits of detection (LOD) and quantitation (LOQ), lin-
earity, inter- and intraday precision, accuracy, and extraction ef-
ficiency.

Table I. MRM Parameters for Paraquat and IS*

Precursor Ion Monitored Ion Collision Energy

Compound (m/z) (m/z) (eV)

PQ 186.00 155.00 35.0

171.00 25.0

IS 214.00 158.00 35.0

185.00 20.0
Figure 1. Structures of paraquat (A) and ethyl viologen (B) * Quantification ions are underlined.

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Journal of Analytical Toxicology, Vol. 35, January/February 2011

The LOD was determined as the lowest concentration of termined by analyzing 1 aliquot of each control over 10 ex-
drug where the calculated value exceeds ±20% of the theoret- tractions. The means of the quantitative results were calculated
ical concentration, but the peak is within ±2% of the calibra- for each concentration. The coefficient of variation (CV) was
tors’ retention time and 20% of the calibrator’s ion ratios. The also calculated, and it is defined as the percentage value of
LOQ is the lowest concentration that meets the described re- the standard deviation divided by the mean. This value must be
quirements with a calculated value within ±20% of the theo- less than 15% for the method to be considered validated per the
retical concentration. laboratory guidelines. The accuracy of the method was calcu-
Selectivity was determined by analyzing six separate sources lated as the percent difference between the theoretical con-
of negative blood for background noise and identifying matrix centration and experimental concentration. This value must be
interferences. Mean area measurements for the selected analyte less than 15% for the method to be considered validated per the
ions were generated and analyzed to establish that the matrices laboratory guidelines.
were free from interfering compounds. Ion suppression and extraction efficiency studies were per-
Five-point calibration curves were used to determine the formed by comparing the unextracted standards of PQ and the
linearity over a concentration range of 2.0–500 ng/mL. The internal standard to extracted blood samples with the com-
target compound and the internal standard peak-area ratios pounds added post- and pre-extraction. The study was per-
were plotted against the concentration, generating a repre- formed at three different concentrations within the linear

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sentative regression analysis.
Intraday precision was performed in one extraction by ana-
lyzing five aliquots of three controls with different concentra-
tions in the established linear range. Interday precision was de-

Table II. Validation for Paraquat

Parameter Value

Linearity* 2.0–500 ng/mL

R2 0.998
Limit of detection 0.5 ng/mL
Limit of quantitation 2.0 ng/mL
Extraction efficiency 93.5%
Precision
Interday† CV < 9.2%
Intraday‡ CV < 2.7%
Accuracy
Interday† 9.7–11.5%
Intraday‡ 6.2–9.0%

* Five-point calibration curve; six extractions. Figure 2. MRM chromatograms of the decendents’ blood containing PQ and
† One aliquot of 3 different concentrations; 10 extractions.
‡ Five aliquots of three different concentrations; one extraction. internal standard.

Table III. Paraquat Concentrations in Fatal Cases

Length of Blood Liver Lung Kidney Stomach Tissue Brain Bile Urine
Sex Age Survival (mg/L) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/L) (mg/L)

male 47 2 days 1.15 1.18

female 20 0–1 day 0.54 11.52
female 18 0–1 day 1.53 9.17 1.37
female 9 0–1 day 17.16 17.75 8.58* 21.28
male† 3 17 days 0.0092 0.0073* 0.037 0.011 0.036 0.0083 0.018

* Only formalin-fixed organ was available.

† Timely treatment with activated charcoal hemoperfusion and dialysis with pulse steroid was attempted with the 3-year-old boy; however, he died after 17 days because of

multiple organ failure.

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Journal of Analytical Toxicology, Vol. 35, January/February 2011

range. The ion suppression was determined to be the signal loss The highly charged PQ is difficult to retain by standard re-
between the unextracted standards and samples where the versed-phase HPLC, however, and alternative approaches are
standards were added post-extraction. The recovery was cal- required. The most widely used are ion-pairing reagents and
culated from the difference between the samples where the highly aqueous conditions (18,19), but ion-pairing reagents
standards were added post-extraction (the ion suppression cause severe ion suppression, reduce the sensitivity, and in-
signal) versus pre-extraction. crease assay variability. For this paper, a hydrophilic-interaction
chromatography (HILIC) column was used. HILIC is a varia-
tion of normal-phase chromatography without the disadvan-
tages of using solvents that are not miscible in water; it could
Results retain highly polar, basic compounds that could not be re-
tained by reversed-phase chromatography. HILIC requires high
The assay proved to be linear with a mean correlation coef- volatility solvents for compound ionization by ESI-MS, which
ficient (n = 10) of 0.998 for PQ. The LOD and LOQ were cal- increases sensitivity compared to the highly aqueous mobile
culated to be 0.5 and 2.0 ng/mL, respectively. Three controls (5, phases used in reversed-phase chromatography. In this study,
50, and 400 ng/mL) within the established linear range were the sensitivity for PQ was increased by utilizing HILIC–ESI-MS
used for the remaining method validation, and the results are with a simple additive in the mobile phase.

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summarized in Table II. In comparison with previously published LC–MS methods in
Interday precision yielded a %CV of less than 9.2% for the combination with SPE, our method shows better precision
three controls. Intraday precision yielded a %CV of less than values, lower analysis time, and similar recovery and lower
2.7%. The method proved to be accurate as the percent differ- limits.
ences between the theoretical value and the experimental value
for the three controls were less than 8.2%.
The total extraction efficiency was determined at three con-
centrations within the linear range. The total percent recovery Conclusions
was above 90%, and the ratio of percent recovery between PQ
and the internal standard behaved similarly during the ex- A simple SPE method using LC–MS instrumentation was de-
traction. More importantly, the ion suppression was minimal veloped to detect and quantitate PQ in biological samples. This
(< 25%) and affected both compounds equally, thus ensuring method allows for accurate measurement of PQ in biological
accurate quantitative results. samples and is practical for use in a forensic laboratory setting.
Table III summarizes the blood and tissue concentrations of The method was validated, and the results proved the method
PQ quantitated with a blood curve for the five positive cases. to be reliable and accurate.
Figure 2 is the chromatogram of the decedents’ blood spec-
imen containing PQ.

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