Abstract:
A method and system for detecting residual poison in human body are provided. Using the disclosed HPLC-Chip-mass spectrometry (MS)/MS and/or HPLC-MS/MS method to detect the residual poison, the method of the present invention mainly includes sample preparation, liquid chromatography and mass spectrometry. The method of the present invention has advantages of low sample size, high specificity, low detection limit, high sensitivity, low cost, high accuracy and stability, etc.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    The present application is a national phase application of the international patent application number PCT/CN2010/078468 filed Nov. 5, 2010 which claims priority from a Chinese invention patent application number 200910221092.5 filed Nov. 5, 2009, and the disclosure of which is incorporated herein by reference and its entirety. 
       TECHNICAL FIELD 
       [0002]    The present invention belongs to the area of biomedical detection technology. In particular, the present invention describes a rapid method and its associated detection system for detecting drug residues in human body. 
       BACKGROUND OF THE INVENTION 
       [0003]    Recent surveys showed an increasing trend of drug abuse, especially in teenager groups of Hong Kong and Shenzhen, and it seems to be spiraling out of control. Drug detection tests would be an important part of the drug control scheme. Blood test and urine test are the common methods in recent decades. The prominent disadvantages include collection complexity, dilution effects, faked sampling problems and short detection periods (less than 7 days). 
         [0004]    Meanwhile, using hair specimens in abused drug detection would be preferred as supplemental evidence for determination of drug use. The first advantage of using hair as a specimen is that it could show the drug-taking history up to 1 year if the hair is longer than 12 cm. Second, embarrassment is avoided during collection of hair samples. It is therefore believed that hair drug tests would be a good alternative to using urine samples. 
         [0005]    Ketamine (“K” as street name) is currently used as veterinary and human anesthetic. Ketamine is classified as an NMDA receptor antagonist. When ketamine is used as a recreational drug, it induces a “dissociative” status of the abuser (mind “separates” from the body). Ketamine abusers are often unconscious and unable to respond to physical stimulation. Furthermore, ketamine has damaging effects cardiovascular, respiratory and nervous systems. 
         [0006]    Methadone is an artificial opiate derivate which has been used for the treatment of dependency of opiates such as heroin and morphine. Clinically, methadone is used to relieve severe chronic pain due to its long lasting action. Methadone is associated with respiratory depression, decreases in heart rate and blood pressure. Individuals who abuse methadone become tolerant and physically depend on methadone during withdrawal treatment. 
         [0007]    Opiates include mono-acetylmorphine, morphine, codeine and heroin. Morphine is the abundant alkaloid found in opium and is a potent opiate analgesic compound to relieve severe pain. Morphine abuser can rapidly develop addiction, tolerance and psychological dependence. 
         [0008]    Heroin (diacetylmorphine) is morphine alkaloid. Its street name is called “white powder”. Heroin is produced from acetylation of morphine. It is typically used as an analgesic drug to treat severe pain. Long term administration causes tolerance, physical dependence and hence addiction. Drug use by injection could cause risk in spreading of blood-bome disease such as AIDS. Heroin is rapidly metabolized into 6-monoacetylmorphine and morphine once administered. 6-monoacetylmorphine is targeted to monitor for abuse. 
         [0009]    Cocaine is a powerful stimulant of the central nervous system and a topical anesthetic. Cocaine increases alertness, euphoria, energy and motor activity. Regular use of cocaine results to psychological dependence and addiction. Because cocaine is usually metabolized into the major metabolite benzoylecgonine (BE), BE is also included in the drug test of cocaine. 
         [0010]    Amphetamines includes amphetamine, methamphetamine, MDMA and MDA. Amphetamine is a psycho-stimulant and produce wakefulness and medically used in the association with fatigue. Amphetamine has been limited to prescription use by FDA. The side effects of amphetamine use are mental fatigue, mental depression. 
         [0011]    Methamphetamine is a psycho-stimulant of the amphetamine class of psychoactive drugs. Its street name often called “ice” or “speed” because of its crystal appearance. Methamphetamine has high potential for abuse and addiction. Its effects include hyperactivity, hallucinations and reduction in appetite. Long term use of methamphetamine associates with depression, suicide, violent behaviors and psychosis. 
         [0012]    MDMA (3,4-methylenedioxy-N-methylamphetamine) belongs to amphetamine class of drug. It is widely known as “ecstasy”. The psychoactive properties of MDMA induce euphoria, increased energy, hyperactive, increased motivation, sexual assault and violent behaviors. MDMA will be metabolized into MDA (3,4-methylenedioxyamphetamine). MDMA and MDA are often quantified in the drug test in blood and urine. 
       SUMMARY OF THE INVENTION 
       [0013]    The first aspect of the present invention relates to a method for detecting drug residues in human hair, urine, seat and oral secretion by incorporating a novel hair sampling scheme and High Performance Liquid Chromatography-Chip-Mass Spectrometry (HPLC-Chip-MS/MS) technology with high sensitivity and reliability. The method of the present invention mainly includes: (1) Sample preparation; (2) Liquid Chromatography; and (3) Mass Spectrometry. Specific conditions of (2) liquid chromatography may include:
       i. Capillary pump having the following conditions: Mobile phase A (0.1-0.2% formic acid in water); Mobile phase B (0.1-0.2% formic acid in acetonitrile); Flow rate: 1-6 μL/min; Mobile phase A gradient (10-97%); Mobile phase B gradient (90-3%); or   ii. Nano pump having the following conditions: Mobile phase A and B are the same to that of the capillary pump; Flow rate: 0.1-0.6 μL/min; Mobile phase A gradient (5-97%); Mobile phase B gradient (95-3%); and   iii. Chip injection volume: 2-4 μL.       
 
         [0017]    Specific conditions of (3) mass spectrometry (MS) may include: Drying gas temperature: 325° C.; Drying gas flow: 4 L/min; Capillary voltage: 1950V; Polarity: Positive ion mode; Scan mode: Multiple Reaction Monitoring (MRM); Ion pair detection: two parent-product ions detection (parameters are referred to respective target drugs). 
         [0018]    The target drugs that the method of the present invention is capable of detecting include ketamine, methadone, morphine, 6-acetylmorphine (metabolite of heroin), cocaine, benzoylecgonine (metabolite of cocaine), amphetamine, methamphetamine, MDMA and MDA. The ion pair used in the method of the present invention for each of these drugs includes:
       i. Ketamine: 238.1&gt;128.0, 238.1&gt;89.0   ii. Methadone: 310.2&gt;265.1, 310.2&gt;91.0   iii. Morphine: 286.1&gt;128.1, 286.1&gt;115.0   iv. 6-acetylmorphine (metabolite of heroin): 328.0&gt;165.0, 328.0&gt;211.0   v. Cocaine: 304.2&gt;105.0, 304.2&gt;182.1   vi. Benzoylecgonine (metabolite of cocaine): 290.1&gt;168.0, 290.1&gt;105   vii. Amphetamine: 136.1&gt;91.0, 136.1&gt;119.0   viii. Methamphetamine: 150.1&gt;65.0, 150.1&gt;91.0   ix. MDMA: 194.1&gt;163.0, 194.1&gt;105   x. MDA: 180.1&gt;135.0, 180.1&gt;163.0       
 
         [0029]    The second aspect of the present invention relates to a method for detecting drug residues in human hair, urine, sweat and oral secretion by incorporating a novel hair sampling scheme and High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS/MS) technology. The method of the present invention mainly includes: (1) Sample preparation; (2) Liquid chromatography; and (3) Mass spectrometry. Specific conditions of (2) liquid chromatography may include: (i) Capillary pump conditions: Mobile phase A (0.1-0.2% formic acid in water); Mobile phase B (0.1-0.2% formic acid in acetonitrile); Flow rate: 0.1-0.6 mL/min; Mobile phase A gradient (10-97%); Mobile phase B gradient (90-3%). Specific conditions of (3) mass spectrometry (MS) may include: Drying gas temperature: 325° C.; Drying gas flow: 10 L/min; Capillary voltage: 4000V; Polarity: Positive ion mode; Scan mode: Multiple Reaction Monitoring (MRM); Ion pair detection: two parent-product ions detection (parameters are referred to respective target drugs). 
         [0030]    The target drugs that the method of the present invention is capable of detecting include ketamine, methadone, morphine, 6-acetylmorphine (metabolite of heroin), cocaine, benzoylecgonine (metabolite of cocaine), amphetamine, methamphetamine, MDMA and MDA. The ion pair used in the method of the present invention for some of these drugs includes:
       i. Ketamine: 238.1&gt;125.0, 238.1&gt;89.0;   ii. Methadone: 310.2&gt;265.1, 310.2&gt;91.0;   iii. Amphetamine: 136.1&gt;91.0, 136.1&gt;119.0;   iv. Methamphetamine : 150.1&gt;65 .0, 150.1&gt;119.0       
 
         [0035]    The human hair that the methods of the present invention are capable of detecting the drug residues therein include head hair, armpit hair, pubic hair and beard hair. 
         [0036]    In an embodiment of detecting the drug residues in human hair, the (1) sample preparation of the methods of the present invention includes:
       i. Collecting the human hair at different lengths;   ii. Washing the collected hair sample in 0.2% SDS, deionized water and acetone by ultrasonication;   iii. Drying by nitrogen gas, 0.5 mL hydrochloric acid (0.15 mol/L) is added for digestion for 4 hours at 60° C. water bath;   iv. Cooling to room temperature, 0.03 mL sodium hydroxide (2 mol/L) and 2 mL sodium phosphate buffer (0.1 mol/L, pH 6.8) are added for neutralization;   v. 2 mL of extraction mixture (90:10 v/v, dichloromethane:hexane) is added for phase extraction by vortexing for 5 minutes;   vi. The sample is taken for centrifugation;   vii. The lower organic phase is collected and dried by nitrogen gas;   viii. The sample is resuspended with 50 μL of acetonitrile;   ix. 5 μL of the resuspended sample is used for liquid chromatography.       
 
         [0046]    The present invention incorporates HPLC-CHIP-MS/MS and HPLC-MS/MS technology in the detection of drug residues. (HPLC-CHIP-MS/MS method was adopted for the detection of commonly abused drugs, viz cocaine, heroin, MDMA/Ecstasy, amphetamine, morphine, ketamine, methadone and methamphetamine. A system according to the HPLC-CHIP-MS/MS method of the present invention is also disclosed in the present invention, which mainly adopts a microfluidic chip-based technology to create a nanospray LC/MS. HPLC-Chip technology is also adopted in the system of the present invention to achieve sample analysis at a very low concentration. Such technology provides higher sensitivity, chromatographic performance, reproducibility, and stability than the traditional HPLC or GC (gas chromatography). The HPLC-Chip in the system of the present invention has a close-channel design to reduce connections, leakage and dead-volume. The advantages of this design include an enhanced sensitivity, a decrease in sample amount and the time for analysis. The detection sensitivity of the system of the present invention can be up to pico-gram level (pg), which is suitable for detecting a trace amount of drugs in a hair sample for as long as 90 days. Accordingly, the number of hair strands collected for detection of drug residues is significantly reduced to only few strands of hair for analysis. The methods are more sensitive than the existing ones, and also give a lower testing cost on average and much shorter testing time. 
     
    
     
       BRIEF DESCRIPTION OF FIGURES 
         [0047]      FIG. 1 : HPLC-Chip-MS/MS Ketamine MRM Spectrum. (A) MRM spectrum from blank hair sample spiked with ketamine standard (20 pg/mg). (B) Calibration curve from blank hair sample spiked with ketamine standard. (C). MRM spectrum from blank hair sample spiked with ketamine standard at the limit of quantification (LOQ) (1 pg/mg). (D) MRM spectrum from blank hair sample spiked with ketamine standard at the limit of detection (LOD) (0.1 pg/mg). 
           [0048]      FIG. 2 : HPLC-Chip-MS/MS Analysis in Hair of Ketamine User. (A) Total ion count chromatogram of ketamine user hair sample. (B) MRM chromatogram of ketamine user hair sample. (C) Product ion MRM chromatogram of ketamine user hair sample. 
           [0049]      FIG. 3 : HPLC-Chip-MS/MS Methadone MRM Spectrum. (A) MRM spectrum from blank hair sample spiked with methadone standard (20 pg/mg). (B) Calibration curve from blank hair sample spiked with methadone standard. (C). MRM spectrum from blank hair sample spiked with methadone standard at the limit of quantification (LOQ) (0.5 pg/mg). (D) MRM spectrum from blank hair sample spiked with methadone standard at the limit of detection (LOD) (0.1 pg/mg). 
           [0050]      FIG. 4 : HPLC-Chip-MS/MS Analysis in Hair of Methadone User. (A) Total ion count chromatogram of methadone user hair sample. (B) MRM chromatogram of methadone user hair sample. (C) Product ion MRM chromatogram of methadone user hair sample. 
           [0051]      FIG. 5 : HPLC-Chip-MS/MS Morphine MRM Spectrum. (A) MRM spectrum from blank hair sample spiked with morphine standard (20 pg/mg). (B) Calibration curve from blank hair sample spiked with morphine standard. (C) MRM spectrum from blank hair sample spiked with morphine standard at the limit of quantification (LOQ) (10 pg/mg). (D) MRM spectrum from blank hair sample spiked with morphine standard at the limit of detection (LOD) (2 pg/mg). 
           [0052]      FIG. 6 : HPLC-Chip-MS/MS Analysis in Hair of Morphine User. (A) Total ion count chromatogram of morphine user hair sample. (B) MRM chromatogram of morphine user hair sample. (C) Product ion MRM chromatogram of morphine user hair sample. 
           [0053]      FIG. 7 : HPLC-Chip-MS/MS 6-acetylmorphine MRM Spectrum. (A) MRM spectrum from blank hair sample spiked with 6-acetylmorphine standard (20 pg/mg). (B) Calibration curve from blank hair sample spiked with 6-acetylmorphine standard. (C) MRM spectrum from blank hair sample spiked with 6-acetylmorphine standard at the limit of quantification (LOQ) (10 pg/mg). (D) MRM spectrum from blank hair sample spiked with 6-acetylmorphine standard at the limit of detection (LOD) (2 pg/mg). 
           [0054]      FIG. 8 : HPLC-Chip-MS/MS Analysis in Hair of 6-acetylmorphine User. (A) Total ion count chromatogram of 6-acetylmorphine user hair sample. (B) MRM chromatogram of 6-acetylmorphine user hair sample. (C) Product ion MRM chromatogram of 6-acetylmorphine user hair sample. 
           [0055]      FIG. 9 : HPLC-Chip-MS/MS Cocaine MRM Spectrum. (A) MRM spectrum from blank hair sample spiked with cocaine standard (20 pg/mg). (B) Calibration curve from blank hair sample spiked with cocaine standard (C) MRM spectrum from blank hair sample spiked with cocaine standard at the limit of quantification (LOQ) (0.5 pg/mg). (D) MRM spectrum from blank hair sample spiked with cocaine standard at the limit of detection (LOD) (0.1 pg/mg). 
           [0056]      FIG. 10 : HPLC-Chip-MS/MS Analysis in Hair of Cocaine User. (A) Total ion count chromatogram of cocaine user hair sample. (B) MRM chromatogram of cocaine user hair sample. (C) Product ion MRM chromatogram of cocaine user hair sample. 
           [0057]      FIG. 11 : HPLC-Chip-MS/MS Benzoylecgonine MRM Spectrum. (A) MRM spectrum from blank hair sample spiked with benzoylecgonine standard (20 pg/mg). (B) Calibration curve from blank hair sample spiked with benzoylecgonine standard. (C) MRM spectrum from blank hair sample spiked with benzoylecgonine standard at the limit of quantification (LOQ) (0.5 pg/mg). (D) MRM spectrum from blank hair sample spiked with benzoylecgonine standard at the limit of detection (LOD) (0.1 pg/mg). 
           [0058]      FIG. 12 : HPLC-Chip-MS/MS Analysis in Hair of Benzoylecgonine User. (A) Total ion count chromatogram of benzoylecgonine user hair sample. (B) MRM chromatogram of benzoylecgonine user hair sample. (C) Product ion MRM chromatogram of benzoylecgonine user hair sample. 
           [0059]      FIG. 13 : HPLC-Chip-MS/MS Amphetamine MRM Spectrum. (A) MRM spectrum from blank hair sample spiked with amphetamine standard (20 pg/mg). (B) Calibration curve from blank hair sample spiked with amphetamine standard. (C) MRM spectrum from blank hair sample spiked with amphetamine standard at the limit of quantification (LOQ) (5 pg/mg). (D) MRM spectrum from blank hair sample spiked with amphetamine standard at the limit of detection (LOD) (2.5 pg/mg). 
           [0060]      FIG. 14 : HPLC-Chip-MS/MS Analysis in Hair of Amphetamine User. (A) Total ion count chromatogram of amphetamine user hair sample. (B) MRM chromatogram of amphetamine user hair sample. (C) Product ion MRM chromatogram of amphetamine user hair sample. 
           [0061]      FIG. 15 : HPLC-Chip-MS/MS Methamphetamine MRM Spectrum. (A). MRM spectrum from blank hair sample spiked with methamphetamine standard (20 pg/mg). (B) Calibration curve from blank hair sample spiked with methamphetamine standard. (C) MRM spectrum from blank hair sample spiked with methamphetamine standard at the limit of quantification (LOQ) (5 pg/mg). (D) MRM spectrum from blank hair sample spiked with methamphetamine standard at the limit of detection (LOD) (2.5 pg/mg). 
           [0062]      FIG. 16 : HPLC-Chip-MS/MS Analysis in Hair of Methamphetamine User. (A) Total ion count chromatogram of methamphetamine user hair sample. (B) MRM chromatogram of methamphetamine user hair sample. (C) Product ion MRM chromatogram of methamphetamine user hair sample. 
           [0063]      FIG. 17 : HPLC-Chip-MS/MS MDMA MRM Spectrum. (A) MRM spectrum from blank hair sample spiked with MDMA standard (20 pg/mg). (B) Calibration curve from blank hair sample spiked with MDMA standard. (C) MRM spectrum from blank hair sample spiked with MDMA standard at the limit of quantification (LOQ) (5 pg/mg). (D) MRM spectrum from blank hair sample spiked with MDMA standard at the limit of detection (LOD) (2.5 pg/mg). 
           [0064]      FIG. 18 : HPLC-Chip-MS/MS Analysis in Hair of MDMA User. (A) Total ion count chromatogram of MDMA user hair sample. (B) MRM chromatogram of MDMA user hair sample. (C) Product ion MRM chromatogram of MDMA user hair sample. 
           [0065]      FIG. 19 : HPLC-Chip-MS/MS MDA MRM Spectrum. (A) MRM spectrum from blank hair sample spiked with MDA standard (20 pg/mg). (B) Calibration curve from blank hair sample spiked with MDA standard. (C) MRM spectrum from blank hair sample spiked with MDA standard at the limit of quantification (LOQ) (5 pg/mg). (D) MRM spectrum from blank hair sample spiked with MDA standard at the limit of detection (LOD) (2.5 pg/mg). 
           [0066]      FIG. 20 : HPLC-Chip-MS/MS Analysis in Hair of MDA User. (A) Total ion count chromatogram of MDA user hair sample. (B) MRM chromatogram of MDA user hair sample. (C) Product ion MRM chromatogram of MDA user hair sample. 
           [0067]      FIG. 21 : HPLC-MS/MS Ketamine MRM Spectrum. (A) MRM spectrum from blank hair sample spiked with ketamine standard (500 pg/mg). (B) Calibration curve from blank hair sample spiked with ketamine standard. (C). MRM spectrum from blank hair sample spiked with ketamine standard at the limit of quantification (LOQ). (D) MRM spectrum from blank hair sample spiked with ketamine standard at the limit of detection (LOD). 
           [0068]      FIG. 22 : HPLC-MS/MS Analysis in Hair of Ketamine User. (A) Total ion count chromatogram of ketamine user hair sample. (B) MRM chromatogram of ketamine user hair sample. (C) Product ion MRM chromatogram of ketamine user hair sample. 
           [0069]      FIG. 23 : HPLC-MS/MS Methadone MRM Spectrum. (A) MRM spectrum from blank hair sample spiked with methadone standard (500 pg/mg). (B) Calibration curve from blank hair sample spiked with methadone standard. (C). MRM spectrum from blank hair sample spiked with methadone standard at the limit of quantification (LOQ). (D) MRM spectrum from blank hair sample spiked with methadone standard at the limit of detection (LOD). 
           [0070]      FIG. 24 : HPLC-MS/MS Analysis in Hair of Methadone User. (A) Total ion count chromatogram of methadone user hair sample. (B) MRM chromatogram of methadone user hair sample. (C) Product ion MRM chromatogram of methadone user hair sample. 
           [0071]      FIG. 25 : HPLC-Chip-MS/MS Amphetamine MRM Spectrum. (A) MRM spectrum from blank hair sample spiked with amphetamine standard (500 pg/mg). (B) Calibration curve from blank hair sample spiked with amphetamine standard. (C) MRM spectrum from blank hair sample spiked with amphetamine standard at the limit of quantification (LOQ). (D) MRM spectrum from blank hair sample spiked with amphetamine standard at the limit of detection (LOD). 
           [0072]      FIG. 26 : HPLC-MS/MS Analysis in Hair of Amphetamine User. (A) Total ion count chromatogram of amphetamine user hair sample. (B) MRM chromatogram of amphetamine user hair sample. (C) Product ion MRM chromatogram of amphetamine user hair sample. 
           [0073]      FIG. 27 : HPLC-MS/MS Methamphetamine MRM Spectrum. (A). MRM spectrum from blank hair sample spiked with methamphetamine standard (500 pg/mg). (B) Calibration curve from blank hair sample spiked with methamphetamine standard. (C) MRM spectrum from blank hair sample spiked with methamphetamine standard at the limit of quantification (LOQ). (D) MRM spectrum from blank hair sample spiked with methamphetamine standard at the limit of detection (LOD). 
           [0074]      FIG. 28 : HPLC-MS/MS Analysis in Hair of Methamphetamine User. (A) Total ion count chromatogram of methamphetamine user hair sample. (B) MRM chromatogram of methamphetamine user hair sample. (C) Product ion MRM chromatogram of methamphetamine user hair sample. 
           [0075]      FIG. 29 : The change of methamphetamine concentration in different hair length of drug abuser. 
           [0076]      FIG. 30 : The change of methadone concentration in different hair length of drug abuser. 
           [0077]      FIG. 31 : The flow chart of HPLC-Chip-MS/MS detection method for drug residues in human hair. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0078]    The practical examples using the methods of the present invention are described as below: 
       Example 1: Use of HPLC-Chip-MS/MS Method in Ketamine Detection 
       [0079]    The procedures for the detection of ketamine in human hair by the HPLC-Chip-MS/MS method of the present invention are as follows: 
         [0080]    (1) Sample preparation 
         [0081]    1. Hair was washed in 0.2% SDS, deionized water and acetone by ultrasonication. After drying by nitrogen gas, 0.5 mL hydrochloric acid (0.15 mol/L) was added for digestion for 4 hours at 60° C. water bath. 
         [0082]    2. After cooling to room temperature, 0.03 mL sodium hydroxide (2 mol/L) and 2mL sodium phosphate buffer (0.1 mol/L, pH 6.8) were added for neutralization. 
         [0083]    3. 2 mL of extraction mixture (90:10 v/v, dichloromethane:hexane) was added for phase extraction by vortexing for 5 minutes. Then, the sample was taken for centrifugation. 
         [0084]    4. The lower organic phase was collected and dried by nitrogen gas. Then, sample was resuspended with 50 μL of acetonitrile. 5 μL was used for liquid chromatography. 
         [0085]    (2) Liquid Chromatography Conditions: 
         [0086]    1. Instruments: 
         [0087]    Agilent 1200 Series LC (Agilent Technologies, Waldbronn, Germany); Analytical column: Agilent chip Zorbax 80SB-C18, 5 μm (Separation: 150 mm×75 μm, Enrichment: 25 mm, 500 nl); Chip is directly installed on the ion source with a micro-camera for monitoring of ionization spray. Chip cube includes chip holder for loading and ejecting chip, valve stator for solvent switching, linkage to micro-plate autosampler with capillary tube and nano electro-ionization spray for ionization. Data acquisition and analysis are performed by Mass Hunter ChemStation Softeare (version B01.03). 
         [0088]    2. Capillary Pump Conditions: 
         [0089]    Mobile phase A (0.1% formic acid in water); Mobile phase B (0.1% formic acid in acetonitrile); Injection volume: 2 μL; Flow rate: 1-6 μL/min (4 μL/min is preferred); Gradient: 0 min (3% B), 3 min (90% B), 5 min (90% B), 5.1 min (3% B), 25 min (3% B). 
         [0090]    3. Nano Pump Conditions: 
         [0091]    Mobile phase A (0.1% formic acid in water); Mobile phase B (0.1% formic acid in acetonitrile); Flow rate: 0.1-0.6 μL/min (0.4 μL/min is preferred); Gradient: 0 min (3% B), 3 min (3% B), 5 min (95% B), 12 min (95% B), 12.1 min (3% B), 25 min (3% B). 
         [0092]    4. Chip Cube Conditions: Injection Flushing Volume (4 μL) 
         [0093]    (3) Mass Spectrometry (MS) Conditions:
   Instruments: Agilent QQQ 6410A   Drying gas temperature: 325° C.   Drying gas flow: 4 L/min   Capillary voltage: 1950V   Polarity: Positive ion mode   Scan mode: Multiple Reaction Monitoring (MRM)   Product ion detection: 238.1&gt;128.0, 238.1&gt;89.0   
 
         [0101]    The detection of ketamine by the HPLC-Chip-MS/MS method is illustrated in  FIGS. 1 and 2 .  FIG. 1  showed the MRM spectrum from blank hair sample spiked with ketamine standard, the calibration curve from blank hair sample spiked with ketamine standard, the limit of quantification (LOQ) and the limit of detection (LOD).  FIG. 2  showed the chromatograms of hair analysis of ketamine by the method of the present invention. The presence of ketamine in the hair sample was shown by the presence of ketamine chromatographic peaks. 
       Example 2: Use of HPLC-Chip-MS/MS Method in Methadone Detection 
       [0102]    Same sample preparation, liquid chromatography conditions and mass spectrometry (MS) conditions as Example 1 are used in this example (except product ion detection). The product ion detection used in this example is 310.2&gt;265.1, 310.2&gt;91.0 
         [0103]    The detection of methadone by the HPLC-Chip-MS/MS method is illustrated in  FIGS. 3 and 4 .  FIG. 3  showed the MRM spectrum from blank hair sample spiked with methadone standard, the calibration curve from blank hair sample spiked with methadone standard, the limit of quantification (LOQ) and the limit of detection (LOD).  FIG. 4  showed the chromatograms of hair analysis of methadone by the method of the present invention. The presence of methadone in the hair sample was shown by the presence of methadone chromatographic peaks. 
       Example 3: Use of HPLC-Chip-MS/MS Method in Morphine Detection 
       [0104]    Same sample preparation, liquid chromatography conditions and mass spectrometry (MS) conditions as Example 1 are used in this example (except injection flushing volume and product ion detection). The injection flushing volume used in this example is 2 μL; the product ion detection used in this example is 286.1&gt;128.1, 286.1&gt;115.0 
         [0105]    The detection of morphine by the HPLC-Chip-MS/MS method is illustrated in  FIGS. 5 and 6 .  FIG. 5  showed the MRM spectrum from blank hair sample spiked with morphine standard, the calibration curve from blank hair sample spiked with morphine standard, the limit of quantification (LOQ) and the limit of detection (LOD).  FIG. 6  showed the chromatograms of hair analysis of morphine by the method of the present invention. The presence of morphine in the hair sample was shown by the presence of morphine chromatographic peaks. 
       Example 4: Use of HPLC-Chip-MS/MS Method in 6-acetylmorphine Detection 
       [0106]    Same sample preparation, liquid chromatography conditions and mass spectrometry (MS) conditions as Example 1 are used in this example (except injection flushing volume and product ion detection). The injection flushing volume used in this example is 2 μL; the product ion detection is 328.0&gt;165.0, 328.0&gt;211.0 
         [0107]    The detection of 6-acetylmorphine by HPLC-Chip-MS/MS is illustrated in  FIGS. 7 and 8 .  FIG. 7  showed the MRM spectrum from blank hair sample spiked with 6-acetylmorphine standard, the calibration curve from blank hair sample spiked with 6-acetylmorphine standard, the limit of quantification (LOQ) and the limit of detection (LOD).  FIG. 8  showed the chromatograms of hair analysis of 6-acetylmorphine by the method of the present invention. The presence of 6-acetylmorphine in the hair sample was shown by the presence of 6-acetylmorphine chromatographic peaks. 
       Example 5: Use of HPLC-Chip-MS/MS Method in Cocaine Detection 
       [0108]    Same sample preparation, liquid chromatography conditions and mass spectrometry (MS) conditions as Example 1 are used in this example (except product ion detection). The product ion detection used in this example is 304.2&gt;105.0, 304.2&gt;182.1 
         [0109]    The detection of cocaine by the HPLC-Chip-MS/MS method is illustrated in  FIGS. 9 and 10 .  FIG. 9  showed the MRM spectrum from blank hair sample spiked with cocaine standard, the calibration curve from blank hair sample spiked with cocaine standard, the limit of quantification (LOQ) and the limit of detection (LOD).  FIG. 10  showed the chromatograms of hair analysis of cocaine by the method of the present invention. The presence of cocaine in the hair sample was shown by the presence of cocaine chromatographic peaks. 
       Example 6: Use of HPLC-Chip-MS/MS Method in Benzoylecgonine Detection 
       [0110]    Same sample preparation, liquid chromatography conditions and mass spectrometry (MS) conditions as Example 1 are used in this example (except product ion detection). The product ion detection used in this example is 290.1&gt;168.0, 290.1&gt;105 
         [0111]    The detection of benzoylecgonine by the HPLC-Chip-MS/MS method is illustrated in  FIGS. 11 and 12 .  FIG. 11  showed the MRM spectrum from blank hair sample spiked with benzoylecgonine standard, the calibration curve from blank hair sample spiked with benzoylecgonine standard, the limit of quantification (LOQ) and the limit of detection (LOD).  FIG. 12  showed the chromatograms of hair analysis of benzoylecgonine by the method of the present invention. The presence of benzoylecgonine in the hair sample was shown by the presence of benzoylecgonine chromatographic peaks. 
       Example 7: Use of HPLC-Chip-MS/MS Method in Amphetamine Detection 
       [0112]    Same sample preparation and mass spectrometry (MS) conditions as Example 1 are used in this example (except liquid chromatography conditions and product ion detection). 
         [0113]    The liquid chromatography conditions used in this example include: 
         [0114]    1. Instruments: 
         [0115]    Agilent 1200 Series LC (Agilent Technologies, Waldbronn, Germany); Analytical column: Agilent chip Zorbax 80SB-Aq, 3.5 μm (Separation: 150 mm×75 μm, Enrichment: 9 mm, 160 nl); Chip is directly installed on the ion source with a micro-camera for monitoring of ionization spray. Chip cube includes chip holder for loading and ejecting chip, valve stator for solvent switching, linkage to micro-plate autosampler with capillary tube and nano electro-ionization spray for ionization. Data acquisition and analysis are performed by Mass Hunter ChemStation Softeare (version B01.03). 
         [0116]    2. Capillary Pump Conditions: 
         [0117]    Mobile phase A (0.1% formic acid in water); Mobile phase B (0.1% formic acid in acetonitrile); Injection volume: 2 μL; Flow rate: 1-6 μL/min (4 μL/min is preferred); Gradient: 0 min (3% B), 3 min (90% B), 5 min (90% B), 5.1 min (3% B), 25 min (3% B). 
         [0118]    3. Nano Pump Conditions: 
         [0119]    Mobile phase A (0.1% formic acid in water); Mobile phase B (0.1% formic acid in acetonitrile); Flow rate: 0.1-0.6 μL/min (0.4 μL/min is preferred); Gradient: 0 min (3% B), 3 min (3% B), 5 min (95% B), 12 min (95% B), 12.1 min (3% B), 25 min (3% B). 
         [0120]    4. Chip Cube Conditions: Injection Flushing Volume (4 μL) 
         [0121]    The product ion detection used in this example is 136.1&gt;91.0, 136.1&gt;119.0. 
         [0122]    The detection of amphetamine by the HPLC-Chip-MS/MS method is illustrated in  FIGS. 13 and 14 .  FIG. 13  showed the MRM spectrum from blank hair sample spiked with amphetamine standard, the calibration curve from blank hair sample spiked with amphetamine standard, the limit of quantification (LOQ) and the limit of detection (LOD).  FIG. 14  showed the chromatograms of hair analysis of amphetamine by the method of the present invention. The presence of amphetamine in the hair sample was shown by the presence of amphetamine chromatographic peaks. 
       Example 8: Use of HPLC-Chip-MS/MS Method in Methamphetamine Detection 
       [0123]    Same sample preparation, liquid chromatography conditions and mass spectrometry (MS) conditions as Example 7 are used in this example (except product ion detection). The product ion detection used in this example is 150.1&gt;65.0, 150.1&gt;91.0 
         [0124]    The detection of methamphetamine by the HPLC-Chip-MS/MS method is illustrated in  FIGS. 15 and 16 .  FIG. 15  showed the MRM spectrum from blank hair sample spiked with methamphetamine standard, the calibration curve from blank hair sample spiked with methamphetamine standard, the limit of quantification (LOQ) and the limit of detection (LOD).  FIG. 16  showed the chromatograms of hair analysis of methamphetamine by the method of the present invention. The presence of methamphetamine in the hair sample was shown by the presence of methamphetamine chromatographic peaks. 
       Example 9: Use of HPLC-Chip-MS/MS Method in MDMA Detection 
       [0125]    Same sample preparation, liquid chromatography conditions and mass spectrometry (MS) conditions as Example 7 are used in this example (except product ion detection). The product ion detection used in this example is 194.1&gt;163.0, 194.1&gt;105 
         [0126]    The detection of MDMA by the HPLC-Chip-MS/MS method is illustrated in  FIGS. 17 and 18 .  FIG. 17  showed the MRM spectrum from blank hair sample spiked with MDMA standard, the calibration curve from blank hair sample spiked with MDMA standard, the limit of quantification (LOQ) and the limit of detection (LOD).  FIG. 18  showed the chromatograms of hair analysis of MDMA by the method of the present invention. The presence of MDMA in the hair sample was shown by the presence of MDMA chromatographic peaks. 
       Example 10: Use of HPLC-Chip-MS/MS Method in MDA Detection 
       [0127]    Same sample preparation, liquid chromatography conditions and mass spectrometry (MS) conditions as Example 7 are used in this example(except product ion detection). The product ion detection used in this example is 180.1&gt;135.0, 180.1&gt;163 .0 
         [0128]    The detection of MDA by the HPLC-Chip-MS/MS method is illustrated in  FIGS. 19 and 20 .  FIG. 19  showed the MRM spectrum from blank hair sample spiked with MDA standard, the calibration curve from blank hair sample spiked with MDA standard, the limit of quantification (LOQ) and the limit of detection (LOD).  FIG. 20  showed the chromatograms of hair analysis of MDA by the method of the present invention. The presence of MDA in the hair sample was shown by the presence of MDA chromatographic peaks. 
       Example 11: Comparison of Conventional GC-MS/MS Method with HPLC-Chip-MS/MS Method on Sensitivity 
       [0129]      
         [0000]    
       
         
               
             
               
               
               
             
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Comparison of Conventional GC-MS/MS with 
               
               
                 HPLC-Chip-MS/MS Method on Sensitivity 
               
             
          
           
               
                   
                 Limit of detection 
                 Limit of quantification 
               
               
                   
                 (LOD) (pg/mg) 
                 (LOQ) (pg/mg) 
               
             
          
           
               
                   
                 Detection method 
               
             
          
           
               
                   
                 HPLC- 
                 GC- 
                 HPLC- 
                 GC- 
               
               
                 Drug 
                 Chip-MS/MS 
                 MS/MS 
                 Chip-MS/MS 
                 MS/MS 
               
               
                   
               
             
          
           
               
                 Ketamine 
                 0.1 
                 200 
                 0.1 
                 500 
               
               
                 Methadone 
                 0.1 
                 200 
                 0.1 
                 800 
               
               
                 Morphine 
                 2 
                 10 
                 2 
                 500 
               
               
                 6-acetyl-morphine 
                 2 
                 30 
                 2 
                 500 
               
               
                 Cocaine 
                 0.1 
                 20 
                 0.1 
                 50 
               
               
                 Benzoylecgonine 
                 0.1 
                 15 
                 0.1 
                 50 
               
               
                 Amphetamine 
                 0.3 
                 160 
                 0.3 
                 500 
               
               
                 Methamphetamine 
                 0.1 
                 100 
                 0.1 
                 500 
               
               
                 MDMA 
                 0.1 
                 200 
                 0.1 
                 500 
               
               
                 MDA 
                 0.5 
                 300 
                 0.5 
                 800 
               
               
                   
               
             
          
         
       
     
       Example 12: Use of HPLC-MS/MS Method in Ketamine Detection 
       [0130]    Sample preparation is the same as that in Example 1. 
         [0131]    Liquid chromatography conditions used in this example include: 
         [0132]    a. Instruments: Agilent 1200 Series LC (Agilent Technologies, Waldbronn, Germany); Analytical column: Agilent ZORBAX Eclipse C18, 3.5 μm, 2.1×100 mm 
         [0133]    b. Capillary Pump Conditions: Flow rate: 0.1-0.6 mL/min (0.4 mL/min is preferred) 
         [0000]    
       
         
               
               
               
             
               
               
               
             
           
               
                   
               
               
                   
                 0.1% formic acid 
                 0.1% formic acid 
               
               
                 Time (min) 
                 in water 
                 in acetonitrile 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 0.0-2.0 
                 95 
                 5 
               
               
                 2.0-2.6 
                 10 
                 13 
               
               
                 2.6-8.0 
                 15.5 
                 84.5 
               
               
                 8.0-8.1 
                 20 
                 80 
               
               
                  8.1-11.0 
                 20 
                 80 
               
               
                   
               
             
          
         
       
     
         [0134]    3. Mass Spectrometry (MS) Conditions: 
         [0135]    Instruments: Agilent QQQ 6410A; Drying gas temperature: 325° C.; Drying gas flow: 10 L/min; Capillary voltage: 4000V; Nebulizer: 35 psi; Polarity: Positive ion mode; Scan mode: Multiple Reaction Monitoring (MRM); Product ion detection: 238.1&gt;125.0, 238.1&gt;89.0 
         [0136]    The detection of ketamine by the HPLC-MS/MS method is illustrated in  FIGS. 21 and 22 .  FIG. 21  showed the MRM spectrum from blank hair sample spiked with ketamine standard, the calibration curve from blank hair sample spiked with ketamine standard, the limit of quantification (LOQ) and the limit of detection (LOD).  FIG. 22  showed the chromatograms of hair analysis of ketamine by the method of the present invention. The presence of ketamine in the hair sample was shown by the presence of ketamine chromatographic peaks. 
       Example 13: Use of HPLC-MS/MS Method in Methadone Detection 
       [0137]    Same sample preparation, liquid chromatography conditions and mass spectrometry (MS) conditions as Example 12 are used in this example (except product ion detection). The product ion detection used in this example is 310.2&gt;265.1, 310.2&gt;91.0 
         [0138]    The detection of methadone by the HPLC-MS/MS method is illustrated in  FIGS. 23 and 24 .  FIG. 23  showed the MRM spectrum from blank hair sample spiked with methadone standard, the calibration curve from blank hair sample spiked with methadone standard, the limit of quantification (LOQ) and the limit of detection (LOD).  FIG. 24  showed the chromatograms of hair analysis of methadone by the method of the present invention. The presence of methadone in the hair sample was shown by the presence of methadone chromatographic peaks. 
       Example 14: Use of HPLC-MS/MS Method in Amphetamine Detection 
       [0139]    Same sample preparation, liquid chromatography conditions and mass spectrometry (MS) conditions as Example 12 are used in this example (except product ion detection). The product ion detection used in this example is 136.1&gt;91.0, 136.1&gt;119.0 
         [0140]    The detection of amphetamine by the HPLC-MS/MS method is illustrated in  FIGS. 25 and 26 .  FIG. 25  showed the MRM spectrum from blank hair sample spiked with amphetamine standard, the calibration curve from blank hair sample spiked with amphetamine standard, the limit of quantification (LOQ) and the limit of detection (LOD).  FIG. 26  showed the chromatograms of hair analysis of amphetamine by the method of the present invention. The presence of amphetamine in the hair sample was shown by the presence of amphetamine chromatographic peaks. 
       Example 15: Use of HPLC-MS/MS Method in Methamphetamine Detection 
       [0141]    Same sample preparation, liquid chromatography conditions and mass spectrometry (MS) conditions as Example 12 are used in this example (except product ion detection). The product ion detection used in this example is 150.1&gt;65.0, 150.1&gt;119.0 
         [0142]    The detection of methamphetamine by the HPLC-MS/MS method is illustrated in  FIGS. 27 and 28 .  FIG. 27  showed the MRM spectrum from blank hair sample spiked with methamphetamine standard, the calibration curve from blank hair sample spiked with methamphetamine standard, the limit of quantification (LOQ) and the limit of detection (LOD).  FIG. 28  showed the chromatograms of hair analysis of methamphetamine by the method of the present invention. The presence of methamphetamine in the hair sample was shown by the presence of methamphetamine chromatographic peaks. 
       Example 16: Hair Analysis in Blind Samples 
       [0143]    Blind test is a crucial examination step to the competency of the method of the present invention. With the application of different conditions in Examples 1-8, ketamine, methadone, morphine, heroin, cocaine, benzoylecgonine, amphetamine and methamphetamine were analyzed in eight blind samples. Table 2 showed that the HPLC-Chip-Ms/MS method can be used to differentiate drug users from non-drug users, and identify the type of drug used. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Blind test results 
               
             
          
           
               
                 Test 
                 Drug abuser ? 
                 Type of drug 
                   
                 Heroin 
                   
                   
                   
               
               
                 no. 
                 (Y/N) 
                 claimed* 
                 Morphine 
                 (6-acetylmorphine) 
                 Amphetamine 
                 Methamphetamine 
                 Methadone 
               
               
                   
               
               
                 1 
                 Y 
                 amphetamines 
                   
                   
                 + 
                 + 
                   
               
               
                 2 
                 N 
               
               
                 3 
                 Y 
                 heroin, methadone 
                   
                 + 
                   
                   
                 + 
               
               
                 4 
                 N 
               
               
                 5 
                 N 
               
               
                 6 
                 Y 
                 heroin, methadone 
                 + 
                 + 
                   
                 + 
                 + 
               
               
                 7 
                 Y 
                 amphetamines, 
                   
                   
                 + 
                 + 
               
               
                   
                   
                 methamphetamine 
               
               
                 8 
                 N 
               
               
                   
               
               
                 (*Provided by Shenzhen Detoxification and Rehabilitation Center) 
               
             
          
         
       
     
       Example 17: Drug Analysis of Abuser in Different Hair Segments 
       [0144]    Parent drug and its metabolites normally appear in hair after 5-7 day once administered. Hair normally grows at 0.75-1 cm/month. Drug taking habit or history can be reflected by analysis in different hair segments. 
         [0145]    Hair specimens were collected and cut into different lengths. With the application of different conditions in Examples 1-8, drug distribution in different hair segments was illustrated ( FIGS. 29 and 30 ). Tables 3 and 4, and  FIGS. 29 and 30  illustrated the drug taking history of methamphetamine and methadone from the target drugs detected in different hair segments which the different lengths of the collected hair segments are proportional to the time history of drug user. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Proportional Relationship between Hair Length and Time History 
               
             
          
           
               
                   
                 Hair segment sample no. 
               
             
          
           
               
                   
                 1 
                 2 
                 3 
                 4 
                 5 
                 6 
                 7 
                 8 
               
               
                   
                   
               
             
          
           
               
                 Hair length (cm) 
                 0-4 
                 4-8 
                 8-12 
                 12-16 
                 16-20 
                 20-24 
                 24-28 
                 28-32 
               
               
                 Time (month) 
                 0-3 
                 3-6 
                 6-9  
                  9-12 
                 12-15 
                 15-18 
                 18-21 
                 21-24 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Detection of Methamphetamine and Methadone Concentration in 
               
               
                 Different Hair Segments 
               
             
          
           
               
                   
                 Hair segment sample no. 
               
             
          
           
               
                   
                 1 
                 2 
                 3 
                 4 
                 5 
                 6 
                 7 
                 8 
               
               
                   
                   
               
             
          
           
               
                 Metham- 
                 117.6 
                 263.6 
                 198.2 
                 201.1 
                 107.3 
                 920 
                 156.3 
                 273.5 
               
               
                 phetamine 
               
               
                 (pg/mg) 
               
               
                 Methadone 
                 18.7 
                 18.3 
                 18.4 
                 18.3 
                 18.3 
                 18.3 
                 18.3 
                 18.4 
               
               
                 (pg/mg) 
               
               
                   
               
             
          
         
       
     
       Example 18: Detection of Drug Residues by HPLC-Chip-MS/MS Method in Urine Sample of Drug Abuser 
       [0146]    (1) Sample Preparation: 
         [0147]    The pH of urine sample was adjusted to pH 9-10 by adding 1 mol/L of NaOH. Sample was loaded on GDX403 column for solid phase extraction. Sample was eluted with dichloromethane. After dryness, sample was resuspended by 50 μL of acetonitrile for analysis. 
         [0148]    (2) Liquid Chromatography Conditions: 
         [0149]    (i) Instruments: 
         [0150]    Agilent 1200 Series LC (Agilent Technologies, Waldbronn, Germany); Analytical column: Agilent chip Zorbax 80SB-C18, 5 μm (Separation: 150 mm×75 μm, Enrichment: 25 mm, 500 nl); Chip is directly installed on the ion source with a micro-camera for monitoring of ionization spray. Chip cube includes chip holder for loading and ejecting chip, valve stator for solvent switching, linkage to micro-plate autosampler with capillary tube and nano electro-ionization spray for ionization. Data acquisition and analysis are performed by Mass Hunter ChemStation Softeare (version B01.03). 
         [0151]    (ii) Capillary Pump Conditions: 
         [0152]    Mobile phase A (0.1% formic acid in water); Mobile phase B (0.1% formic acid in acetonitrile); Injection volume: 2 μL; Flow rate: 1-6 μL/min (4 μL/min is preferred); Gradient: 0 min (3% B), 3 min (90% B), 5 min (90% B), 5.1 min (3% B), 25 min (3% B). 
         [0153]    (iii) Nano Pump Conditions: 
         [0154]    Mobile phase A (0.1% formic acid in water); Mobile phase B (0.1% formic acid in acetonitrile); Flow rate: 0.1-0.6 μL/min (0.4 μL/min is preferred); Gradient: 0 min (3% B), 3 min (3% B), 5 min (95% B), 12 min (95% B), 12.1 min (3% B), 25 min (3% B). 
         [0155]    4. Chip Cube Conditions: Injection Flushing Volume (2-4 μL) 
         [0156]    (3) Mass Spectrometry (MS) Conditions:
   Instruments: Agilent QQQ 6410A; Drying gas temperature: 325° C.; Drying gas flow:   4 L/min; Capillary voltage: 1950V ; Polarity: Positive ion mode; Scan mode: Multiple   Reaction Monitoring (MRM)   
 
         [0160]    The ion pair used in the method of the present invention for detecting some of these drugs in the urine samples includes:
       i. Ketamine: 238.1&gt;128.0, 238.1&gt;89.0;   ii. Methadone: 310.2&gt;265.1, 310.2&gt;91.0;   iii. Morphine: 286.1&gt;128.1, 286.1&gt;115.0;   iv. 6-acetylmorphine (metabolite of heroin):328.0&gt;165.0, 328.0&gt;211.0;   v. Cocaine: 304.2&gt;105.0, 304.2&gt;182.1;   vi. Benzoylecgonine (metabolite of cocaine): 290.1&gt;168.0, 290.1&gt;105;   vii. Amphetamine: 136.1&gt;91.0, 136.1&gt;119.0;   viii. Methamphetamine: 150.1&gt;65.0, 150.1&gt;91.0;   ix. MDMA: 194.1&gt;163.0, 194.1&gt;105;   x. MDA: 180.1&gt;135.0, 180.1&gt;163.0       
 
       Example 19: Detection of Drug Residues by HPLC-Chip-MS/MS Method in Oral Secretion of Drug Abuser 
       [0171]    (1) Sample Preparation: 
         [0172]    Ethyl acetate was added to the oral secretion samples. Then the samples were treated with liquid-liquid extraction by ultrasonication for 2 mins. Layer of ethyl acetate was collected and concentrated. The concentrated samples were resuspended by 50 μL of acetonitrile for analysis. 
         [0173]    (2) Liquid Chromatography Conditions and (3) Mass Spectrometry Conditions are the Same as Example 18. 
       Example 20: Detection of Drug Residues by HPLC-Chip-MS/MS Method in Sweat of Drug Abuser 
       [0174]    (1) Sample Preparation: 
         [0175]    Filter paper was put under the armpit of the subject. Sweat was collected for 20 mins in a 35° C. temperature control room. The filter paper was then cut into small pieces. Dichloromethane was added and shake thoroughly. After brief centrifugation, the dichloromethane layer was collected and concentrated. The concentrated sample was resuspended by 50 μL of acetonitrile for analysis. 
         [0176]    (2) Liquid Chromatography Conditions and (3) Mass Spectrometry Conditions are the Same as Example 18 
         [0177]    If desired, the different functions discussed herein may be performed in a different order and/or concurrently with each other. Furthermore, if desired, one or more of the above-described functions may be optional or may be combined 
         [0178]    Although various aspects of the invention are set out in the independent claims, other aspects of the invention comprise other combinations of features from the described embodiments and/or the dependent claims with the features of the independent claims, and not solely the combinations explicitly set out in the claims. 
         [0179]    It is also noted herein that while the above describes exemplary embodiments of the invention, these descriptions should not be viewed in a limiting sense. Rather, there are several variations and modifications which may be made without departing from the scope of the present invention as defined in the appended claims. 
       INDUSTRIAL APPLICATION 
       [0180]    The present invention can fulfill different demands of drug testing. The methods of the present invention only require lesser specimen amount but are highly specific and sensitive, low in cost, accurate and reliable. The present invention can be applied by government executive agency, inspection units and pharmaceutical industry.