Mol Cell Toxicol (2011) 7:185-189 DOI 10.1007/s13273-011-0025-x
ORIGINAL PAPER
Application of liquid chromatography tandem mass spectrometry for the simultaneous quantification of multiple non-opioid drugs in human plasma Kwang-Youl Kim1,*, Ju-Hee Kang1,3,*, Cheol-Woo Kim1,2 & Moonsuk Nam1,2
Received: 20 October 2010 / Accepted: 18 May 2011 �The Korean Society of Toxicogenomics and Toxicoporeteomics and Springer 2011
Abstract The simultaneous, quantitative and rapid analysis of plasma concentrations of multiple drugs is important to determine the clinical decision and to expect the prognosis in patients administered in emergency unit with intoxication. Here, we developed the liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based drug screening method for analyzing 12 drugs (acetaminophen, amitriptyline, chlorpromazine, cimetidine, diazepam, doxylamine, ephedrine, imipramine, metoclopramide, propranolol, tramadol, and zolpidem) with frequent events of intoxication throughout the country and evaluated its clinical applicability. The overall sensitivity (low limit of quantitation, 0.1-0.5 μg/mL), specificity, precision and accuracy for the quantification of 12 drugs were reliable and all drugs can be analyzed within 6 min. Among 12 drugs in samples for quality control, the REMEDi HS-based method detected only 6 drugs with low accuracy, while the LC-MS/MS system was able to precisely quantify all drugs. In addition, pilot analysis of patient samples with unknown drug intoxication was superior to the conventional LC-based drug profiling system, and was rapid and cost effective. In conclusion, LC-MS/MS-based drug screening is a good replacement for conventional LC-based REMEDi analyzer and has the better clinical applicability. 1
Clinical Trial Center, Inha University Hospital, Incheon, Korea Department of Internal Medicine, School of Medicine, Inha University, Incheon, Korea 3 Department of Pharmacology and Medicinal Toxicology Research Center, Research Institute for Medical Sciences, Center for Advanced Medical Education by BK-21 Project, School of Medicine, Inha University, Incheon, Korea *These authors contributed equally to this work Correspondence and requests for materials should be addressed to M. Nam (
[email protected]) 2
Keywords Liquid chromatography mass spectrometry, LC-MS/MS, REMEDi analyzer, Drug screening The first priority of the treatment of the poisoned patients arrived in emergency department is clinical stabilization1,2. Once the poisoned patients is clinically stabilized, it should be estimated the level of exposure to the poison. However, particularly in the case of intentional self-poisoning, it may be difficult to collect the information about poisons in numerous cases. The quantitative determination of plasma concentration of overdosed drug can often provide both prognostic and therapeutic guidance. For example, the plasma concentration of salicylate or acetaminophen in overdosed patient is useful to determine the therapeutic strategy using nomogram3. However, if health providers fail to obtain the information about poisoned drugs or chemicals, the appropriate quantitative analysis method for the determination of unknown materials will be great useful. Several tens of toxins or drugs are commonly poisoned in human. The characteristics of poisons are different according to the country, culture, socioeconomic status, or urbanization. For instance, intoxication by organophosphorus insecticides and herbicides are relatively common in rural area, whereas abused opioids or other clinical drugs are common intoxicants in urban region. The liquid chromatography with ultraviolet detection (LC/UV) platform for screening of poisoned drugs in biologic samples was widely used for several decades4. However, there are many limitations are present, including sensitivity, time-consuming of sample preparation, chromatographic separation, and lack of quantification. Therefore, the more efficient and precise method to
186
Mol Cell Toxicol (2011) 7:185-189
Intensity (cps)
screening of multiple drugs commonly poisoned should be developed. Here, we presented the more efficient and sensitive method using LC tandem mass spectrometry (LC-MS/MS) for the quantification and screen3.2e5 3.0e5 2.8e5 2.6e5 2.4e5 2.2e5 2.0e5 1.8e5 1.6e5 1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e4 0.0
ing of 12 commonly intoxicated drugs in human plasma as an alternative to the LC/UV platform high throughput drug profiling system (REMEDi).
6 1. Cimetidine 2. Ephedrine 3. Acetaminophen 4. Metoclopramide 5. Tramadol 6. Doxylamine 7. Zolpidem 8. Propranolol 9. Imipramine 10. Amitryptyline 11. Chlorpromazine 12. Reserpine (Int. Std.) 13. Diazepam
5 4 3 9 12 2
8 10
1
0.5
11 13
7
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
Time (min)
Figure 1. Representative chromatogram of 12 drugs (1 μg/mL) spiked with blank plasma. Reserpine was used as an internal standard (Int. Std.) with concentration of 20 μg/mL.
Table 1. Validation data for linearity, precision and accuracy of QC samples. Analyzed drugs
RT (min)
Linearity (μg/mL)
r2
QC (μg/mL)
Mean (μg/mL)
Accuracy (%)
[RSD] (%)
0.28 8.70
94 109
6.4 8.0
AAP
1.12
0.1-10
0.9989
0.3 8
AMIT
3.00
0.2-10
0.9996
0.3 8
0.26 8.49
86.9 106
14.9 5.8
CPZ
3.42
0.5-10
0.9985
8
7.89
98.7
1.4
CMT
0.93
0.1-10
0.9983
0.3 8
0.30 7.61
99.3 95.2
0.7 5.1
DZP
5.10
0.1-10
0.9996
0.3 8
0.289 7.11
96.4 88.8
3.8 12.5
DOX
1.34
0.1-10
0.9985
0.3 8
0.317 7.25
106 90.7
5.4 10.3
EPH
1.00
0.1-10
0.9995
0.3 8
0.291 8.43
97 105
3.1 5.1
IMP
2.70
0.2-10
0.9997
0.3 8
0.273 8.6
90.9 107
9.9 7.0
MCP
1.18
0.1-10
0.9989
0.3 8
0.309 7.08
103 88.5
2.9 13.0
PRO
1.68
0.1-10
0.9974
0.3 8
0.281 7.19
93.5 89.8
6.8 11.3
TMD
1.29
0.1-10
0.9982
0.3 8
0.31 7.25
103 90.7
3.2 10.3
ZPM
1.47
0.1-10
0.9992
0.3 8
0.35 7.4
117 92.5
14.3 8.1
Abbreviations: AAP, acetaminophen; AMIT, amitriptyline; CPZ, chlorpromazine; CMT, cimetidine; DZP, diazepam; DOX, doxylamine; EPH, ephedrine; IMP, imipramine; MCP, metoclopramide; PRO, propranolol; TMD, tramadol; ZPM, zolpidem
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Specificity
Representative chromatograms of drug-free plasma and plasma spiked with 12 drugs are shown in Figure 1. In blank plasma, we could not observe any interfering peak from endogenous compounds at the retention times of the each analyzed drug or internal standard from different lots (data not shown). In addition, no late-eluting peak was observed and new injection could be done in every 6 min. Total of analyzed drugs could be detected within 5.5 min (from less than 1 min to 5.1 min; Table 1 and Figure 1). Sensitivity and linearity
The linearity was investigated by constructing the calibration curves from plasma spiked with drugs at six
points of level from 0.1 to 10 μg/mL. Triplicate analyses were performed for each calibration to evaluate the repeatability. The calibration curves of all drugs showed good linearity over the analysis range. As shown in Table 1, the low limit of quantification (LLOQ) defined as the lowest concentration of the analyzed drug with an precision and accuracy within 20% was as low as 0.1 μg/mL (except amitriptyline and imipramine, 0.2 μg/mL; chlorpromazine, 0.5 μg/mL). Validation of the LC-MS/MS-based drug screening method and clinical application
As shown in Table 2, the intra-day and inter-day precision and accuracy acquired from samples for quality control (concentrations ranged from 0.3 to 8 μg/mL)
Table 2. Intra-day and inter-day validation of QC samples for 3 days with various concentrations. Analyzed drugs
QC (μg/mL)
Interday
Intraday
Mean (μg/mL)
Accuracy (%)
[RSD] (%)
Mean (μg/mL)
Accuracy (%)
[RSD] (%)
AAP
0.3 4 8
0.3 3.7 7.7
96.3 91.4 95.7
3.6 9.5 4.5
0.3 4.3 8.6
109.0 109.0 108.0
8.3 8.1 7.5
AMIT
0.3 4 8
0.3 3.5 7.8
88.8 86.3 97.4
12.6 14.3 2.7
0.3 3.6 8.5
92.4 91.3 106.3
8.2 9.6 6.3
CPZ
4 8
3.8 7.9
93.9 98.7
6.5 1.4
3.5 7.9
87.0 98.4
14.9 1.6
CMT
0.3 4 8
0.3 3.8 7.8
99.8 94.9 97.0
0.1 5.4 3.1
0.3 3.9 7.7
105.6 97.3 95.9
5.5 2.7 4.3
DZP
0.3 4 8
0.3 3.8 7.8
94.3 95.3 97.9
6.0 4.8 2.2
0.3 4.2 8.4
99.3 104.7 105.0
0.7 4.7 5.0
DOX
0.3 4 8
0.3 4.1 7.7
104.7 103.3 96.7
4.6 3.3 3.3
0.3 4.4 8.7
109.7 110.3 109.0
9.0 9.4 8.1
EPH
0.3 4 8
0.3 4.0 7.9
99.0 99.2 98.2
1.1 0.8 1.9
0.3 4.3 8.4
105.3 108.7 105.3
5.3 8.0 4.9
IMP
0.3 4 8
0.3 3.6 7.9
86.2 90.0 98.2
16.0 11.0 1.7
0.3 4.1 8.7
90.1 102.0 109.0
10.8 2 8.2
MCP
0.3 4 8
0.3 4.1 8.0
102.0 102.0 99.7
2.2 2.1 0.4
0.3 4.7 9.1
110.7 116.7 113.7
9.6 14.5 11.9
PRO
0.3 4 8
0.3 4.1 7.6
101.2 102.3 94.9
1.3 2.2 5.2
0.3 4.6 8.7
106.3 114.7 109.0
5.9 12.9 8.2
TMD
0.3 4 8
0.3 4.2 8.0
104.7 105.9 100.2
4.4 5.7 0.0
0.3 4.7 9.0
115.0 117.3 112.0
13.0 14.4 10.6
ZPM
0.3 4 8
0.3 4.0 8.1
111.0 100.7 101.8
9.9 0.9 1.8
0.3 4.5 9.0
114.3 113.3 112.0
12.7 11.8 10.9
188
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Table 3. Comparison of LC-MS/MS-based quantification of 12 drugs to the REMEDi-based quantification using QC samples with concentration of 1 μg/mL. Analyzed drugs AAP AMIT CPZ CMT DZP DOX EPH IMP MCP PRO TMD ZPM
LC-MS/MS
REMEDi HS
Calculated concentration (μg/mL) 1.13 1.06 1.00 1.08 1.04 1.13 1.10 1.10 1.13 1.11 1.14 1.06
N.D. N.D. N.D. N.D. N.D. 0.64 N.D. N.D. 1.16 0.68 0.90 N.D.
N.D.; Not detected.
for each compound were within 20%. Intra-day precision ranged between 0.7 and 14.9% whereas the interday precision ranged between 0.0 and 16.0%. The intra-day mean error was between -12.09 and 17.3% whereas inter-day mean error was between -13.8 and 11.0%. The mean absolute recovery ratios for each drug measured at concentration of 1 μg/mL using the present extraction procedure were between 58.5 (propranolol) and 107.4% (chlorpromazine). To evaluate the applicability of our LC-MS/MS-based drug screening method as comparing with REMEDi HS system, we measured concentrations of 12 drugs (concentration of 1 μg/mL spiked with blank plasma) using LC-MS/MS and conventional REMEDi HS system (as methods of manufacturer’s instruction). With the REMEDi HS due in most cases to a lack of sensitivity, specificity and co-elution capability were not detected in 6 drugs and also quantification was wrong (¤15%, C.V) about 2 out of 4 drugs, while the LC-MS/MS system was able to precisely quantify all drugs (Table 3).
Discussion We selected drugs to analyze using the LC-MS/MS platform based on the most common intoxicated drugs in selected university hospital. One of the most common intoxicated materials is organophosphorus insecticide in Korea, particularly in rural area5. Fortunately, a simple spectrophotometric method to measure the activity of acetylcholinesterase using red blood cell in peripheral blood reflecting the level of organophosphorus intoxication has been established6. Other drugs listed in the commonly poisoned materials are similar
to the selected drugs in the present study. Therefore, if the valid, specific and rapid method to quantify the exposure levels of drugs is established, clinician can not only be able to expect the therapeutic outcome or prognosis, but also be able to prepare the exact strategy of treatment. Recently, the very sensitive and valid platform to analyze the blood level of various endogenous and exogenous chemicals using mass spectrometry has been introduced7-9. In the connection with this, our system to quantify the most common intoxicated chemicals or drugs will be useful. In addition, a rapid, sensitive and valid method to analyze the biologic samples will contribute to enroll the subjects appropriate to the clinical trial. Here, we reported the rapid, sensitive and valid method based on LC-MS/MS platform, and which will be contribute to select the appropriate subjects for clinical trials who are free of drug interaction between above common medicines and new drug candidate under development. We successfully detected all drugs using small volume of plasma with concentration ranged from 0.1-0.5 μg/mL to 10 μg/mL. In the case of acetaminophen, the hepatotoxic concentration of acetaminophen in the plasma sampled within 1 hr after oral ingestion is over 300 μg/mL3. In the contrast, the plasma concentration of overdosed doxylamine or clozapine is usually within several μg/ mL10,11. Therefore, the method being able to measure the wider range of concentration of drugs is necessary. Our method presented in this study was very rapid and sensitive. Therefore, if necessary, plasma collected in patients with intoxication by unknown drugs can be diluted to several times. To exclude subject contaminated with other drugs in clinical trial, we should sensitively detect various drugs rapidly. Among the drugs analyzed in this study, drugs such as cimetidine, doxylamine and acetaminophen are over-the-counter drugs. Therefore, these drugs can be usually contaminated in subjects enrolled in clinical trial. In addition to drugs analyzed in this study, other drugs with potential of drug interaction should be added in the list for the drug screening in clinical trial setting. In the future, the method could be updated. In addition, using the more robust platform such as QTRAP mass spectrometry, the numerous intoxicated drugs or chemicals will be measured in the patients with intoxication, or in subjects for clinical trials. In conclusion, the LC-MS/MS method described in this study is suitable for the analysis of non-opioid drugs in human plasma and also the calibration curve range of 12 drugs is enough to screen intoxicated patients using a small aliquot of human plasma. This assay has been used to analyze several hundreds of clinical
Mol Cell Toxicol (2011) 7:185-189
samples with a rapid, specific, precise, accurate and ruggedness as demonstrated in the method validation.
189
Acknowledgements This work was supported by a grant of the Korean Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (A070001).
Materials & Methods Materials and preparation of standard solution
Reserpine, acetonitrile, methanol, formic acid and ammonium acetate were HPLC grade and were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals or solvents were of the highest grade and were purchased commercially. Stock standard solution of 12 drugs were dissolved in 50% methanol and working standards were diluted serially with 50% methanol to final concentrations at 1, 2, 5, 10, 50, and 100 μg/mL. Plasma sample preparation
Samples for calibration curves and quality control (QC) were prepared by dilution of each working standard solution with blank human plasma. To twenty μL of plasma sample, same volume of internal standard (20 μg/mL, reserpine) and 1 mL of acetonitrile were added, and then mixed well for 30 seconds by vortex. After centrifugation at 14,000×g for 5 min, 1 μL of supernatant was transferred into auto-sampler for injection into the LC-MS/MS system. Analytical conditions
All drugs were analyzed with an API 4000 MS/MS system (Applied Biosystems, MDS SCIEX, Canada) using multiple reaction monitoring (MRM) method. Data processing was performed with the Analyst 1.5 software (Applied Biosystems). The operating parameters for the ESI source in the positive mode for analysis are follows: curtain gas, 10 psi; CAD gas, 5 psi; ion spray voltage, 4,000 V; temperature, 300� C; DP, 21-81; collision energy, 19-35 V. The chromatographic conditions were as follows: column, Imtakt C8 (3 μm, 75×2.0 mm); flow rate, 0.2 mL/min; mobile phase, A solution (water containing 10 mM ammonium acetate and 0.1% formic acid): B solution (acetonitrile =60 : 40; column temcontaining 0.1% formic acid)= perature, 40� C; injection volume, 1 μL.
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