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Capillary electrophoresis for the analysis of drugs of abuse in biological specimens of forensic interest
Trends in Analytical Chemistry, Vol. 31, 2012
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Capillary electrophoresis for the analysis of drugs of abuse in biological specimens of forensic interest C. Cruces-Blanco, A.M. Garcı´a-Campan˜a We review analytical methodologies using capillary electrophoresis and related techniques (micellar electrokinetic chromatography and capillary electrochromatography) with detection systems (ultraviolet-visible spectrometry, fluorescence, laser-induced fluorescence and mass spectrometry) for quantification of drugs of abuse and their metabolites in biological specimens of interest in forensic toxicology (e.g., blood, urine and hair). Despite some drawbacks that still need to be addressed and finally overcome when using this technique in forensic laboratories, the coupling of capillary electrophoresis and mass spectrometry generally provides a powerful option for detection and determination of very low concentrations of these compounds in some forensic matrices (e.g., hair). ª 2011 Elsevier Ltd. All rights reserved. Keywords: Blood; Capillary electrochromatography; Capillary electrophoresis; Drugs of abuse; Forensic analysis; Hair; Mass spectrometry; Metabolite; Micellar electrokinetic chromatography; Urine C. Cruces-Blanco*, A.M. Garcı´a-Campan˜a Department of Analytical Chemistry, Faculty of Sciences, University of Granada, C/Fuentenueva s/n, E-18071 Granada, Spain
*
Corresponding author. Tel.: +34 958240451; E-mail: [email protected]
1. Introduction The abuse of addictive drugs is increasing world-wide, causing serious social problems, and, as a result, their production, trade and use are strictly regulated [1]. In order to protect human health and to comply with stringent legislation, the development and the application of analytical methods of drugs of abuse in biological specimens are very important [2,3]. These analyses in forensic laboratories have traditionally been carried out using gas chromatography (GC) and highperformance liquid chromatography (HPLC) [4]. GC, especially coupled with mass spectrometry (MS), is the most frequently used reference and confirmation technique for many types of drugs, mostly in urine samples, but, more recently, also in hair samples [5,6]. Its main limitation is that it is not applicable to highly polar, nonvolatile and thermally-unstable substances, including many of the target drugs of forensic interest. In most instances, these
0165-9936/$ - see front matter ª 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2011.06.019
drawbacks can be overcome by introducing a derivatization step, but that introduces a new source of variability and complexity. HPLC, mainly in combination with MS, is widely used for analysis of drugs of abuse in forensic samples, offering simplicity and eliminating the need to carry out a derivatization procedure, thus saving time and resources [7–10]. Capillary electrophoresis (CE) and related techniques are increasingly being employed in forensic analysis. Like HPLC, these methods are also suitable for automation, high sample throughput, high efficiency, much flexibility and low consumption of samples and reagents – which have permitted their application to a wide range of areas, including pharmaceuticals, food and beverages, environmental and clinical analysis [11–16]. Two different modes are used mainly [i.e. capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC)]. Some works have used organic modifiers {e.g., organic solvents in non-aqueous capillary electrophoresis (NACE) mode
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[17]} or other organic substances (e.g., cyclodextrins) used for the separation of enantiomers (CD-CE) [18]. The use of capillary electrochromatography (CEC), which combines the high efficiency of CZE and the high selectivity of HPLC in forensic analysis, is also worth mentioning [19–21]. The potential of CE for forensic analysis was first demonstrated in 1991 by Weinberger and Lurie [22], who applied it to the analysis of a wide range of illicit drugs in synthetic mixtures. Since this first paper, numerous reviews have been published summarizing the works on the forensic applications of CE [23–33]. The exceptional power of separation and resolution, short analysis time, economical use of reagents, and minimum sample requirements make CE an attractive methodology for forensic laboratories [34,35]. In contrast to these advantages, the primary disadvantage limiting its use is its poor concentration sensitivity, particularly when applied with on-column ultraviolet (UV) detection, due to low sampleinjection volume and short optical pathlength. This can be overcome with fluorescence (FL), especially laser-induced FL (LIF), detection. The introduction of MS techniques in the field of forensic toxicology has revolutionized the way in which forensic laboratories have approached many of the analytical problems with which they are confronted. Most of the advantages of MS detection, common to the well-established coupling of HPLC with MS in forensic toxicology [36,37], also appear when CE is coupled with MS. CE-MS is opening great opportunities to gain wider acceptance in forensic toxicology [38–40]. Forensic analysis of drugs of abuse is usually carried out in blood, urine and hair. Although blood and urine are the most common and preferred matrices used for toxicological studies involving drugs of abuse, hair is gaining in importance as an alternative specimen for documentation of use or exposure to drugs, because of its different advantages, especially its stability and ease for sampling and storage, compared with conventional biological samples [41,42]. This review gives an overview of the literature of CE, CE-MS, MEKC and CEC methodologies for drugs of abuse analysis in these three sorts of biological specimen.
2. Sample-preconcentration methods to enhance sensitivity In addition to using the above-mentioned sensitive detectors, two further possibilities are available for increasing the sensitivity of CE [i.e. decreasing the limits of detection (LODs) and quantitation (LOQs)], which include off-line and on-line sample-preconcentration methods. Among the off-line preconcentration methods, liquid-liquid extraction (LLE) or solid-phase extraction (SPE) are often applied to enrich the analytes and get rid 86
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of the sample matrix [43]. However, the whole procedure with multiple steps is time consuming and requires a large amount of organic solvent. Solid-phase microextraction (SPME) was introduced as an alternative preconcentration method, since it is a solventless method that requires a small amount of sample while providing accurate, reproducible results [44]. On-line sample-preconcentration techniques, based on electrophoresis, are the easiest ways to achieve sample enrichment in CE, since the preconcentration step is performed within the same capillary as the analysis [45]. The most frequently used preconcentration methods in the analysis of drugs of abuse by CZE mode are fieldamplified sample stacking (FASS) [46] and large-volume sample stacking (LVSS) [47]. FASS is considered to be the simplest technique for on-line sample concentration. By carrying out proper sample preparation in a low conductivity matrix, filling the capillary with a high-conductivity background electrolyte (BGE) and applying a high positive voltage, to achieve 10–10,000-fold sensitivity enhancements. LVSS is performed using a buffer system similar to that used for FASS, but the electrode polarity is switched so as to acquire a reversed electroosmotic flow (EOF). In the initial step, the sample is dissolved in a low-conductivity buffer or water. After the capillary is filled with a highconductivity BGE, the sample solution is injected into the capillary to a certain length. A negative polarity is applied and the direction of the EOF is toward the inlet. The anionic analytes move toward the detection end (outlet) and stack at one side of the boundary between the sample zone and the BGE, while the cations and neutral species move and exit the capillary at the injection end (inlet). Compared to FASS, this preconcentration method can provide much larger sample injection without any significant loss in separation efficiency, but it cannot be used for the simultaneous separation of anions and cations and, also, it is limited to analytes with low mobilities. Among the most frequently encountered methods for enhancing sensitivity in the analysis of drugs of abuse by the MEKC mode are the normal stacking and sweeping [48], especially combined with cation-selective exhaustive injection (CSEI), the so-called CSEI-sweeping method, which was first reported by Quirino and Terabe [49]. It results in more sensitive detection than sweeping and is sufficiently flexible to offer the potential to achieve an increase in the LOD of more than 100,000 fold, for positively charged analytes. At the beginning of the runs, the capillary is conditioned with a non-micellar BGE, followed by the injection of a high-conductivity buffer and, finally, the injection of a short water plug. Using electrokinetic injection at a positive polarity, the cationic analytes accumulate in a low-conductivity matrix or water. They enter the capillary through the water plug at high velocities and are then focused or stacked at the interface
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between the water zone and the high-conductivity buffer. Once an optimized injection time is determined, it is stopped and the micellar BGEs are replaced at both ends of the capillary. The voltage is then switched to negative polarity, thus permitting the entry of micelles from the cathodic vial into the capillary to sweep the analytes stacked and introduced to the narrow bands. Finally, the separation can be performed using MEKC in reversemigration mode. Compared to conventional off-line sample-preconcentration methods (LLE and SPE), the stacking methods are rather economical, sensitive, rapid, simple and reproducible.
3. Capillary electrophoresis with different detection systems 3.1. UV detection CE-UV coupling is the most widely used CE mode for the analysis of many drugs of abuse in biological samples, mainly urine. This type of detector is standard in commercial instruments, which can also be fitted with a diode-array detector for the simultaneous acquisition of spectra [50] However, the short internal diameter of the capillary (detection pathlength) limits the sensitivity of this detection system, and, consequently, LODs for UV-Vis absorption are usually not low enough for the quantification of drugs of abuse in biological samples. Thus, most of these applications require CE sensitivity to be enhanced by using more sensitive detection systems or by involving on-line or off-line sample-preconcentration methods, as discussed above. One of the first applications was by Wernly and Thormann in 1991 [51], who employed the MEKC mode for the qualitative analysis of different drugs of abuse and/or their metabolites. The peak identification was based on not only migration times, but also the on-line
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recorded UV spectra of the peaks. After SPE of 5 mL of urine, drug concentrations down to the ng/mL level were analyzable in this biological matrix. The same authors [52] showed that, using direct injection of urine, it was possible to achieve an LOD of about 20 lg/mL for the major metabolites of heroin and morphine-3-glucuronide using CZE and MEKC with UV detection. Lower LODs were obtainable when off-line preconcentration by SPE was employed. Highly specific analysis with short sample preparation with SPE and good recovery were obtained for a mixture of two opium alkaloids and two anesthetics in urine [53]. The group of McCord [54] developed and validated a method to screen urine for 19 different drugs of abuse, but the selectivity of the system was limited by the use of UV detection at 214 nm, a wavelength at which many endogenous compounds absorb. To avoid interferences from the matrix, an automated sequential injection SPE provided a preconcentration effect that gave very low LODs. UV detection was also employed at a very low wavelength (190 nm) with CZE and MEKC mode in the presence of SDS, enabling the analysis of morphine in human plasma at therapeutic concentrations [55]. A screening method for opiates, amphetamine and caffeine in urine and serum was reported by Hyo¨tyla¨inen et al. [56] by using MEKC-UV with an analysis time of only 2 min. Also, eight urine samples obtained from individuals undergoing methadone therapy were analyzed by CZE-UV without interference from coextracted drugs of abuse and/or their metabolites [57]. Another CE-UV method was proposed by Brown et al. [58] for the analysis of three common club drugs requiring minimal sample preparation and urine-sample volume with a run time of less than 7 min. Also, different substances related to heroin addiction and treatment were analyzed by CEUV with appropriate LODs [59]. Different amphetamines were analyzed in human whole-blood samples by CE-UV in less than 7 min, without the need of any
Figure 1. Electropherogram of (1) amphetamine, (2) methamphetamine, (3) 3,4-methylenedioxyamphetamine, (4) 3,4-methylenedioxyethyl amphetamine, (5) 3,4-methylenedioxymethamphetamine and (6) 2,5-dimethoxy-4-methyl-phenethylamine with DAD detection at 200 nm. CE conditions: capillary (50 cm x 50 lm internal diameter), temperature, 25C, running solution, 100 mM phosphate buffer (pH 2.5); voltage, 10 kV. With permission from ref. [60].
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derivatization procedure (see Fig. 1) [60], but needing a simple extraction method to obtain very good sensitivity. Coupling of CZE with UV detection was first used for the determination of illicit drugs in hair samples by Tagliaro et al. in 1993 [61]. The extraction procedure, preceded by an acidic incubation for 24 h, attained the LODs required for this type of sample (about 0.2 ng/mg). The same research group carried out a very convenient method with very good recoveries for routine analysis of cocaine in hair by employing CE-DAD [62]. As mentioned above, b-CD can be used to enhance selectivity in enantiomeric resolution being carried out by CE [18]. Qi et al. [63] used a multi-target inmunoaffinity column prior to CE separation with UV detection using b-CD as additive for the determination of morphine and related compounds in urine of heroin abusers obtaining very good reproducibility and precision. The method proposed by Liau et al. [64] also permits the chiral separation of methamphetamine, methacathionone, ephedrine and pseudoephedrine using b-CD in a CE-UV method. The determination of enantiomers in clandestine tablets and urine samples represents a good complementary method to GC-MS for use in forensic and clinical analysis. b-CD was also employed for the simultaneous separation of six drugs of abuse in urine and hair samples by CE-UV after a simple LLE extraction [65]. To increase the sensitivity required to carry out hair analysis, a FASS on-column preconcentration procedure was applied. The same chiral selector, added to a phosphate buffer, also offers excellent resolution and sensitivity for the analysis of human urine and hair samples from 6-acetylmorphine users [66]. A simple work-up procedure based on LLE permits very good recoveries. The simultaneous determination of morphine, codeine, 6-acetylmorphine and normorphine was
also carried out using CE-UV with b-CD in the BGE. The application of electrokinetic injection with FASS resulted in low LODs (40 ng/mL) [67]. To obtain a 1000-fold sensitivity enhancement, the FASS on-column sample-preconcentration procedure was applied to the determination of opioids in urine [68]. The same detection system was used in CE for the analysis of four amphetamines applying electrokinetic injection with FASS in urine samples [69]. Also, Taylor et al. [70] provided an excellent quantitative determination of opiates using a BGE of disodium hydrogen phosphate at neutral pH, obtaining very good results when carrying out the methodology in the same biological matrix. The FASS procedure was also applied to the analysis of hair samples to increase the sensitivity required for this type of biological specimen. The simultaneous analysis of morphine, cocaine and 3,4-methylenedioxymethamphetamine was carried out with CE-UV [71]. Also, the FASS procedure was used for the determination of different major opiates in hair samples, employing acidic BGE in the presence of organic solvent [72]. Four amphetamines in spiked hair samples were also analyzed by CZE using this type of preconcentration procedure that increased detection sensitivity by up to about 1000-fold [73]. The CSEI-sweeping sample-preconcentration procedure, usually employed in MEKC mode, was used for the determination of heroin, morphine, codeine and 6-monoacetylmorphine in urine samples [74]. The analysis was carried out in less than 10 min and only a simple LLE extraction was needed to get rid of salts. Lin et al. [75] developed another CSEI-sweep MEKC method to determine morphine, codeine, normorphine, morphine-3-glucoronide and morphine-6-glucoronide also in urine samples, obtaining a sensitivity increase of 2500-fold with respect to the CZE mode. The same on-
Figure 2. Separation of three amine drugs by using CSEI-sweeping. (1) 3,4-methylenedioxymethamphetamine, (2) methamphetamine (3) amphetamine and (IS) benzylamine at a concentration level of 5 ng/mL. With permission from ref. [76].
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Figure 3. Electrochromatopherograms resulting from the analysis of (a) blank urine sample, (b) urine sample spiked with a standard mixture of: (1) amphetamine, (2) methamphetamine, (3) 3,4-methylenedioxyamphetamine, (4) 3,4-methylenedioxymethamphetamine, (5) 3,4-methylenedioxyethylamphetamine, (6) heroin, (7) codeine, (8) 6-monoacetylmorphine, (9) cocaine and (10) morphine. The sample was electrokinetically injected (7 kV x 10 s); mobile phase, 20 mM phosphate (pH 2.5)/acetonitrile (80/20, v/v). With permission from ref. [21].
column preconcentration procedure was developed for the determination of cocaine and its metabolites in urine [76]. The BGE was a mixture of phosphoric acid, SDS, 2-propanol and tetrahydrofuran, and, using an electrokinetic sample injection, led to 1000-fold reductions in LODs. The applicability of this preconcentration procedure in the analysis of forensic samples was also demonstrated in the determination of methamphetamine, ketamine, codeine and morphine in hair samples employing MEKCUV mode [75]. As seen in Fig. 2, the combination of CSEI with the MEKC mode also lowered by down to 1000-fold the LOD of three amphetamines (3,4-methylenedioxymethamphetamine, methamphetamine, amphetamine and benzylamine) in hair samples using a BGE in presence of SDS and an organic solvent [77]. The CSEI-sweeping sample-preconcentration procedure was also employed for separation and determination of basic compounds of forensic interest in human urine. Fig. 3 shows the electropherograms corresponding to a blank urine sample and spiked samples, treated following the proposed procedure [50]. A very interesting methodology employing a particular type of stacking called low temperature bath (LTB) stacking, coupled with the NACE mode, was used for the analysis of 3,4-methylenedioxymethamphetamine in illicit drug and urine samples [78]. A portion of the capillary was immersed in a low-temperature bath, which served as a ‘‘pseudo-low-conductivity zone’’ and, as a result, a large volume of sample injection could be achieved, lowering the LODs dramatically.
3.2. Fluorescence detection FL-HPLC detection has been widely used in many fields of analytical chemistry with some examples in the field of forensic analysis [23,79,80] because of the sensitivity and the selectivity that this detection system can offer. However, on-column FL detection has sensitivity limitations, due to the difficulties in focusing enough excitation energy into the capillary and efficiently collecting the radiation emitted [23]. In such cases or in those where the analytes do not exhibit strong enough native FL, they can be derivatized with a proper derivatization agent, but the advantage of detecting native FL is that no precolumn-derivatization step is necessary, simplifying the chemical work-up and reducing the time of total analysis. The lack of significant native FL of most of the molecules can be an important advantage of FL detection if selectivity is required. Another way to enhance sensitivity and selectivity greatly is the combination of CE with a LIF detector, requiring the appropriate laser sources. The use of FL/LIF detection in forensic analysis is mainly restricted to urine samples. Methods for separation and determination of normorphine, morphine, 6-acetylmorphine and codeine in biological fluids using CE and LIF detection were described by Alnajjar et al. [81]. When the assay was based on native FL, LODs in the range of 200 ng/ml were achieved. To enhance sensitivity further, a two-step precolumn-derivatization procedure using 1-chloroethyl chloroformate and fluorescein isothiocyanate (FITC) as the reagents was employed before LIF detection, leading to LODs at the 50–100 pg/mL level.
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Phenethylamine designer drugs were determined in urine samples by MEKC-LIF based on their native FL and using LLE and sweeping preconcentration to improve the LODs [82]. Using FITC as derivatization agent before a CELIF method, six illicit drugs (ephedrine, pseudoephedrine, amphetamine, methampetamine, 3,4-methylendioxyamphetamine and 3,4-methylendioxymethylamphetamine) were simultaneously determined with an LOD of 200 pg/mL [83]. 3.3. Mass-spectrometry detection To overcome the drawbacks of UV and FL detection modes discussed above, CE coupled with MS was recently established as a powerful method for forensic screening [84,85]. This technique, with its excellent detection sensitivity and with the special characteristic of providing structural information on the analytes, has become complementary to HPLC-MS [86,87] and is especially useful for the analysis of matrices (e.g., hair) with low drug content. However, due to the high price and limited commercial availability of CE-MS instruments, this technique is not yet in general use in forensic analysis. In CE-MS, the soft ionization techniques are also in use in HPLC-MS. Currently, electrospray ionization (ESI) is the most popular interface between CE and MS, because it facilitates the transfer of analytes from the liquid phase of the CE to the gas phase of the MS. Atmospheric pressure electron-impact (APEI) ionization also gave very good sensitivity and selectivity for a wide range of analytes of clinical and forensic significance [88]. Using
an ESI interface, da Costa et al. determined cocaine and its major metabolites in human urine [89]. The coupling of CE-ESI-MS, preceded by simple SPE, has also enabled the determination of different derivatives of 2,5-dimethylamphetamine [90]. With the same interface, another method was proposed to screen and to quantify four thiophenethylamine designer drugs in human plasma after a simple LLE for sample clean-up [91]. 3,4-methylenedioxy-methamphetamine and methadone were determined using protein precipitation prior to the hydrodynamic injection of the samples [92]. A method has been proposed by Gottardo et al. for the determination of illicit drugs in blood samples after LLE and FASS on-column preconcentration using CZE with ESI and time-of-flight (TOF)-MS [93]. 3,4-methylenedioxymethamphetamine and three of its main metabolites were also analyzed in plasma and urine samples after administration of ecstasy [94]. Ion-trap (IT) MS is also a useful tool for forensic analysis. Confirmation testing of urinary amphetamines and designer drugs by CE/IT-APEI-MS gave very good results [95]. Wey and Thormann [68] also applied a CE-MS with FASS to the determination of opioids in urine samples, resulting in a 1000-fold sensitivity enhancement with respect to UV detection. The same authors [96] used CEMS with APEI for the analysis of morphine and related opioids in the same biological specimen with hydrody-
Table 1. Analytical methodologies for drugs of abuse in blood samples Analytes Morphine
Morphine, heroin, codeine, amphetamine, caffeine Amphetamine, methamphetamine, 3,4methylenedioxyamphetamine, 3,4methylenedioxyethylamphetamine, 3,4methylenedioxymethamphetamine, 2,5dimethoxy-4-methyl-phenethylamine Thiophenethylamine designer drugs (2CT-series) Methamphetamine, 3,4methylenedioxyamphetamine, 3,4methylenedioxyethylamphetamine, 3,4methylenedioxymethamphetamine, methadone, cocaine, benzoylecgonine, morphine, codeine and, 6acetylmorphine) 3,4-methylenedioxymethamphetamine, 3,4-methylelenedioxyamphetamine, 4hydroxy-3-methoxymethamphetamine and 4-hydroxy-3-methoxyamphetamine Ecstasy and methadone
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Experimental conditions
Mode of detection
LOD
Reference
BGE of disodium tetraborate decahydrate (pH 10.5) and SDS (50 mM) 50 mM glycine and 50 mM SDS (pH 10.5) BGE of 100 mM phosphate buffer (pH 2.5)
CZE-UV (190 nm) MEKC-UV
50 ng/mL
[55]
MEKC-UV
0.34–1.20 lg/mL
[56]
CZE-UV (200 nm)
10–30 ng/mL
[60]
LLE
CE-ESI-MS
11.3–23.0 ng/mL
[91]
LLE FASS, ammonium formate (25 mM) (pH 9.5)
CE-ESI-TOF-MS
2–10 ng/mL
[93]
CE-ESI-TOF-MS
2–10 ng/mL
[94]
CE-MS
0–175 ng/mL
[92]
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Table 2. Analytical methodologies for drugs of abuse in urine samples Analytes Benzoylecgonine, morphine, heroin, 6acetylmorphine, methamphetamine, codeine, amphetamine, cocaine, methylephedrine, methaqualone, benzodiazepines Morphine-3-glucoronide Lidocaine, bupivacaine, noscapine, papaverine 19 drugs of abuse Methadone Methamphetamine, 3,4methylenedioxyamphetamine, ketamine Morphine, codeine, methadone, 2-ethylidene1,5-dimethyl-3,3-diphenylpyrrolidine Morphine, codeine, acetylcodeine, 6acetylmorphine, morphine-3-glucoronide 3,4-methylenedioxymethamphetamine, 3,4methylenedioxyamphetamine, ephedrine, amphetamine, methamphetamine Methamphetamine, methacathionone, ephedrine and pseudoephedrine Opioids Amphetamine, methamphetamine, 3,4methylenedioxyamphetamne, 3,4,methylenedioxymethamphetamine Pholcodine, 6-monoacetylmorphine, morphine, heroin, codeine, dihydrocodeine Heroin, morphine, codeine, 6-acetylmorphine Morphine, codeine, normorphine, morphine-3glucoronide, morphine-6-glucuronide Cocaine, benzoylecgonine, norcocaine, cocaethylene Morphine, heroin, codeine, amphetamine, caffeine Amphetamine, methamphetamine, 3,4methylenedioxyamphetamine, 3,4methylenedioxymethamphetamine, cocaine, codeine, heroin, morphine, 6-acetylmorphine 3,4-methylenedioxymethamphetamine 2C-T-2, 2C-T-7, 2c-c. 2c-B, « c-I Normorphine, 6-acetylmorphine, codeine, morphine Pseudoephedrine, ephedrine, amphetamine, methamphetamine, 3,4methylenedioxyamphetamine, 3,4methylenedioxymethamphetamine Normorphine, codeine, 6-acetylmorphine Cocaine, benzoylecgonine, cocaethylene, anhydroecgonine, anhydroecgonine methyl ester, ecgonine methyl ester
2,5-dimethylamphetamine and derivatives
Experimental conditions
Mode of detection
LOD
References
SPE Borate/phosphate buffer (75 mM) with SDS
MEKC-UV
100 ng/mL
[51]
Sample extraction with C8 cartridges SPE BGE with b-CD and organic. SPE Extraction with disposable cartridges
CZE-UV MEKC-UV CZE-UV CE-UV
20 lg/mL 1 lg/mL 300 ng/mL 5–30 ng/mL
[52]
CZE-UV
20 ng/mL
[57]
CE-UV
25–300 lg/mL
[58]
[53] [54]
CE-UV Inmmunoaffinity column and b-CD b-CD and LLE
[59]
CE-UV
10–20 ng/mL
[63]
CE-UV(200 nm) CE-DAD (190–240 nm)
500 ng/mL
[65]
b-CD
CE-UV
FASS
CZE-UV
25–34 lg/mL
[68] [69]
FASS and SPE
CZE-UV
4–9 ng/mL
[70]
CSEI-sweeping and LLE CSEI-sweeping
MEKC-UV MEKC-UV
10 ng/mL 10–35 ng/mL
[74] [75]
CSEI-sweeping
MEKC-UV
–
[76]
50 mM glycine and 50 mM sodium dodecyl sulfate (pH 10.5) SPE on strong cation exchange cartridges
MEKC-UV
0.34–1.20 lg/mL
[56]
CEC-UV
5–12 ng/mL
[21]
Low temperature bath stacking Sweeping and FITC derivatization ACE-CI and FITC
NACE-UV
10
CE-FL MEKC-LIF CE-FL CE-LIF CE-LIF
10 7–10
200 ng/mL 50–100 pg/mL 200 pg/mL
[81]
CE-FL CE-LIF CE-ESI-MS
200 ng/mL 50–100 pg/mL 100–250 ng/mL
[67]
CE-ESI-MS
10–1000 ng/mL [90] (continued on next page)
Derivatization with FITC
Formic acid (1 M) as electrolyte and formic acid (0.05 M) in methanol:water as coaxial sheath liquid SPE
[64]
6
M–5.0 x 10 8
M
9
M
[78] [82]
[83]
[89]
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Table 2. (continued) Analytes 3,4-methylenedioxymethamphetamine, 3,4methylelenedioxyamphetamine, 4-hydroxy-3methoxymethamphetamine, 4-hydroxy-3methoxyamphetamine Amphetamine, methamphetamine, 3,4methylenedioxymethamphetamine, 3,4methyelenedioxyamphetamine Opioids Morphine and related opioids
Experimental conditions
CE-ESI-TOF-MS
BGE of ammonium acetate and acetic acid (pH 4.6) FASS Hydrodynamic sample injection
namic sample injection. The LODs were in the ng/mL level. Zhang et al. recently reported a CE-MS method based on a charged polymer-protected gold nanoparticlecoated capillary for the analysis of heroin and its basic impurities [97]. Although some limitations regarding the selection of CE-electrolyte composition and the concentration sensitivity of the CE-MS coupling have not yet been completely overcome, several applications have been published in the field of drugs-of-abuse analysis in hair [98,99]. Gottardo et al. [98] developed a simple, rapid CZE-IT-MS method for the sensitive, quantitative determination of different drugs of abuse and their metabolites in human hair, applying FASS preconcentration to lower the LODs to the severe cut-offs adopted in this type of analysis (i.e. 0.1 ng/mg) for both clinical and forensic purposes. When coupling CE-ESI-MS and CE-TOF-MS with a simple LLE, off-line preconcentration, the determinations of different drugs in the same matrix at the low concentrations required were also obtained [99]. Another CZE-ESI/TOF-MS method was developed and validated for the identification and determination of numerous drugs of abuse in blood and hair samples [93]. A confirmatory assay based on SPE extraction followed by CE-ESI-MS has been employed for the simultaneous analysis of enantiomers of methamphetamine, amphetamine, 2,5-dimethylamphetamine, ephedrine, norephedrine and methylephedrine hydrochloride in urine samples [100]. The same ionization process was employed for the analysis of human urine [101] after the administration of codeine and codeine6-glucoronide.
4. Applications to biological samples Typically, the biological samples subjected to drug analysis for forensic toxicology purposes are widely different and include almost any tissue and organ, but blood, urine, saliva, hair and nails are most important for detecting drugs of abuse [102]. In order to limit the
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Mode of detection
LOD 2–10 ng/mL
CE-API-MS CE-IT-MS CE-MS CE-MS CE-APEI-MS
References [94]
[95]
100–200 ng/mL
[68] [96]
workload due to the huge increase of casework occurring in recent decades, there is a tendency to limit as much as possible the specimens to be analyzed to those having a fundamental value for the purpose of the investigation [103]. In accordance with this tendency, we have focused our attention in this review to blood, urine and hair, which represent the majority of specimens analyzed for drugs of abuse in most laboratories. 4.1. Analysis of blood samples Plasma and serum are typically used for emergency testing in forensics and toxicology, and in therapeutic monitoring, as they are suitable as samples to determine the short-term use of drugs. Drawbacks are that blood is an invasive sample, difficult to handle and reflects the patientÕs dosages for some hours only. Some examples are shown in Table 1. As seen in Table 1, UV and MS are the two detection systems employed, and, since the sensitivity required in this biological sample is higher than in urine, off-line preconcentration procedures, such as LLE, are applied. 4.2. Analysis of urine samples Urine is the most commonly used biological sample for the analysis of drugs of abuse (e.g., amphetamines and opiates) in forensic laboratories. It is generally used for the detection of these compounds in schools and workplaces, because it is obtainable non-invasively, as compared to blood sampling. The main limitation in its use is that it provides direct evidence only for short-term use of illicit drugs because these remain in urine no longer than for 10 days after use [104]. Also, urine analysis cannot distinguish between habitual and first-time users. Nevertheless, as seen in Table 2, most of the applications of CE methodologies found in the literature are carried out with urine. UV detection generally used does not usually offer the sensitivity required for this type of analysis. For this reason, various off-line and on-line preconcentration procedures are usually employed, alone or in combination (e.g., LLE or SPE with FASS or CSEI-sweeping), resulting in LODs at the ng/mL level.
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Table 3. Analytical methodologies for drugs of abuse in hair samples Analytes Cocaine, morphine
Cocaine Methamphetamine, methacathionone, ephedrine, pseudoephedrine Ephedrine, amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methyelenedioxyamphetamine, 3,4-methylenedioxyethylamphetamine Morphine, cocaine, 3,4-methylenedioxymethamphetamine Morphine, codeine, 6-monoacetylmorphine, acetylcodeine, heroin Amphetamine, methamphetamine, 3,4-methyelenedioxyamphetamine, 3,4-methylenedioxymethamphetamine Methamphetamine, ketamine, codeine, morphine 3,4-methyelenedioxyamphetamine, methamphetamine, amphetamine, benzylamine 6-monoacetylmorphine, morphine, amphetamine, methamphetamine, 3,4-methyelenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, benzoylecgonine, ephedrine, cocaine Amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethaamphetamine ephedrine, cocaine, morphine, codeine, 6-acetylmorphine, benzoylecgonine Methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxyethylamphetamine, 3,4-methylenedioxymethaamphetamine methadone, cocaine, benzoylecgonine, morphine, codeine, 6-acetylmorphine
Experimental conditions
Mode of detection
Acidic incubation overnight in 0.25 M HCl (45C) and extraction with Toxi-tubes 50 cm x 75 lm capillary
LOD
Reference
CZE-UV (214 and 238 nm)
0.15 ng/mg
[61]
CE-DAD (200 to 400 nm)
0.15–0.35 ng/ mg 300 ng/mL
[62] [66]
150 mM phosphate buffer (pH 2.5) + 15 mM b-CD FASS b-CD (15 mM)
CE-UV (200 nm) CE-UV (200 nm) or multiwavelength (190– 400 nm)
0.2 lg/mL
[65]
FASS
CE-UV (200 nm) or multiwavelength (190– 400 nm) CE-UV
2–8 ng/mL
[71]
0.75 ng/mL to 150 pg/mL
[72]
FASS, BGE (sodium phosphate + 40% ethylene glycol (pH 2.5) FASS, BGE (phosphate buffer, 100 mM + 40% ethylene glycol (pH 2.5) CSEI-sweeping
CZE-UV
0.06 lg/mL
[73]
MEKC-UV
50–100 pg/mL
[75]
CSEI-sweeping
MEKC-UV
0.01 lg/mL to 50 pg/mL
[77]
FASS
CE-IT-MS
0.1 ng/mg
[98]
LLE
CE-ESI-MS CE-TOF-MS
0.1 ng/mg
[99]
FASS LLE
CZE-ESI-MS CZE-TOF-MS
0.1 ng/mg
[93]
Using FL (especially LIF) detection, LODs can be decreased to the pg/mL level. But because most of the drugs of abuse under study do not present native FL, a previous derivatization step with a derivatization reagent, usually FITC, is often required, complicating the analysis methodology. Due to its advantages discussed in sub-section 3.3, CE-MS is becoming the most desirable method in forensic laboratories. 4.3. Analysis of hair samples Undoubtedly, urine and blood are the routine samples of choice for drug analysis, but, beside them, hair is being recognized as the third fundamental biological specimen for drug testing [105]. Although more than 450 papers dealing with hair analysis for drugs have been published
since 1954, most of them have appeared in this decade [106]. One of the reasons for this increase is that hair can provide a historical account of drug intake or exposure, whereas fluid matrices (e.g., the previously cited urine or blood) can give information about only fairly recent drug use [107]. Hair analysis is currently applied in workplace drug testing, post-mortem toxicology, doping control and certification of physical fitness to drive vehicles. Also, it has been used to investigate ancient peoplesÕ lives (e.g., diet and cause of death) in archaeological studies [108], but its maximum potential is in forensic toxicological and drugs of abuse studies. The use of hair in forensic toxicology was introduced in the late 1970s by Baumgartner et. al. [109], and,
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from the analytical toxicology point of view, it poses specific problems, especially sample collection, preparation and extraction. Nevertheless, it has demonstrated many advantages compared with blood and urine. As a sample, it is a obtainable non-invasively, it is easy to preserve and it is the only one that provides information about the quantity and historic pattern of drug use, having a retrospective period of months or years, saving drugs and their metabolites unchanged. The reason is that drugs enter the hair stalk at the hair root together with the nutrients from the capillary blood and remain embedded in the of hair matrix for all the life of this structure (from months to years). Since the rate of growth of hair is about 1 cm per month, each cm of hair keeps the record of about 1 month of use or exposure of the individual to drugs. This type of information is not available from any other specimen. Only a limited number of papers have described methodologies for hair analysis based on CE (see Table 3). The contribution of the group of Tagliaro from the University of Verona is remarkable [61,62,65, 66,71]. The detection system most frequently employed, at present, is UV, which does not give the required sensitivity in this type of biological matrix. For this reason, it is usually necessary to apply a sample-preconcentration procedure, FASS being employed most often. However, the most recently published methods for the analysis of drugs of abuse in hair employed MS as the detection system [10,93,99], so CE-MS will be the most important technique for this type of analysis in forensic laboratories in the near future. References [1] [2] [3] [4]
[5] [6] [7] [8]
[9]
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[14]
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Capillary electrophoresis for the analysis of drugs of abuse in biological specimens of forensic interest C. Cruces-Blanco, A.M. Garcı´a-Campan˜a We review analytical methodologies using capillary electrophoresis and related techniques (micellar electrokinetic chromatography and capillary electrochromatography) with detection systems (ultraviolet-visible spectrometry, fluorescence, laser-induced fluorescence and mass spectrometry) for quantification of drugs of abuse and their metabolites in biological specimens of interest in forensic toxicology (e.g., blood, urine and hair). Despite some drawbacks that still need to be addressed and finally overcome when using this technique in forensic laboratories, the coupling of capillary electrophoresis and mass spectrometry generally provides a powerful option for detection and determination of very low concentrations of these compounds in some forensic matrices (e.g., hair). ª 2011 Elsevier Ltd. All rights reserved. Keywords: Blood; Capillary electrochromatography; Capillary electrophoresis; Drugs of abuse; Forensic analysis; Hair; Mass spectrometry; Metabolite; Micellar electrokinetic chromatography; Urine C. Cruces-Blanco*, A.M. Garcı´a-Campan˜a Department of Analytical Chemistry, Faculty of Sciences, University of Granada, C/Fuentenueva s/n, E-18071 Granada, Spain
*
Corresponding author. Tel.: +34 958240451; E-mail: [email protected]
1. Introduction The abuse of addictive drugs is increasing world-wide, causing serious social problems, and, as a result, their production, trade and use are strictly regulated [1]. In order to protect human health and to comply with stringent legislation, the development and the application of analytical methods of drugs of abuse in biological specimens are very important [2,3]. These analyses in forensic laboratories have traditionally been carried out using gas chromatography (GC) and highperformance liquid chromatography (HPLC) [4]. GC, especially coupled with mass spectrometry (MS), is the most frequently used reference and confirmation technique for many types of drugs, mostly in urine samples, but, more recently, also in hair samples [5,6]. Its main limitation is that it is not applicable to highly polar, nonvolatile and thermally-unstable substances, including many of the target drugs of forensic interest. In most instances, these
0165-9936/$ - see front matter ª 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2011.06.019
drawbacks can be overcome by introducing a derivatization step, but that introduces a new source of variability and complexity. HPLC, mainly in combination with MS, is widely used for analysis of drugs of abuse in forensic samples, offering simplicity and eliminating the need to carry out a derivatization procedure, thus saving time and resources [7–10]. Capillary electrophoresis (CE) and related techniques are increasingly being employed in forensic analysis. Like HPLC, these methods are also suitable for automation, high sample throughput, high efficiency, much flexibility and low consumption of samples and reagents – which have permitted their application to a wide range of areas, including pharmaceuticals, food and beverages, environmental and clinical analysis [11–16]. Two different modes are used mainly [i.e. capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC)]. Some works have used organic modifiers {e.g., organic solvents in non-aqueous capillary electrophoresis (NACE) mode
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[17]} or other organic substances (e.g., cyclodextrins) used for the separation of enantiomers (CD-CE) [18]. The use of capillary electrochromatography (CEC), which combines the high efficiency of CZE and the high selectivity of HPLC in forensic analysis, is also worth mentioning [19–21]. The potential of CE for forensic analysis was first demonstrated in 1991 by Weinberger and Lurie [22], who applied it to the analysis of a wide range of illicit drugs in synthetic mixtures. Since this first paper, numerous reviews have been published summarizing the works on the forensic applications of CE [23–33]. The exceptional power of separation and resolution, short analysis time, economical use of reagents, and minimum sample requirements make CE an attractive methodology for forensic laboratories [34,35]. In contrast to these advantages, the primary disadvantage limiting its use is its poor concentration sensitivity, particularly when applied with on-column ultraviolet (UV) detection, due to low sampleinjection volume and short optical pathlength. This can be overcome with fluorescence (FL), especially laser-induced FL (LIF), detection. The introduction of MS techniques in the field of forensic toxicology has revolutionized the way in which forensic laboratories have approached many of the analytical problems with which they are confronted. Most of the advantages of MS detection, common to the well-established coupling of HPLC with MS in forensic toxicology [36,37], also appear when CE is coupled with MS. CE-MS is opening great opportunities to gain wider acceptance in forensic toxicology [38–40]. Forensic analysis of drugs of abuse is usually carried out in blood, urine and hair. Although blood and urine are the most common and preferred matrices used for toxicological studies involving drugs of abuse, hair is gaining in importance as an alternative specimen for documentation of use or exposure to drugs, because of its different advantages, especially its stability and ease for sampling and storage, compared with conventional biological samples [41,42]. This review gives an overview of the literature of CE, CE-MS, MEKC and CEC methodologies for drugs of abuse analysis in these three sorts of biological specimen.
2. Sample-preconcentration methods to enhance sensitivity In addition to using the above-mentioned sensitive detectors, two further possibilities are available for increasing the sensitivity of CE [i.e. decreasing the limits of detection (LODs) and quantitation (LOQs)], which include off-line and on-line sample-preconcentration methods. Among the off-line preconcentration methods, liquid-liquid extraction (LLE) or solid-phase extraction (SPE) are often applied to enrich the analytes and get rid 86
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of the sample matrix [43]. However, the whole procedure with multiple steps is time consuming and requires a large amount of organic solvent. Solid-phase microextraction (SPME) was introduced as an alternative preconcentration method, since it is a solventless method that requires a small amount of sample while providing accurate, reproducible results [44]. On-line sample-preconcentration techniques, based on electrophoresis, are the easiest ways to achieve sample enrichment in CE, since the preconcentration step is performed within the same capillary as the analysis [45]. The most frequently used preconcentration methods in the analysis of drugs of abuse by CZE mode are fieldamplified sample stacking (FASS) [46] and large-volume sample stacking (LVSS) [47]. FASS is considered to be the simplest technique for on-line sample concentration. By carrying out proper sample preparation in a low conductivity matrix, filling the capillary with a high-conductivity background electrolyte (BGE) and applying a high positive voltage, to achieve 10–10,000-fold sensitivity enhancements. LVSS is performed using a buffer system similar to that used for FASS, but the electrode polarity is switched so as to acquire a reversed electroosmotic flow (EOF). In the initial step, the sample is dissolved in a low-conductivity buffer or water. After the capillary is filled with a highconductivity BGE, the sample solution is injected into the capillary to a certain length. A negative polarity is applied and the direction of the EOF is toward the inlet. The anionic analytes move toward the detection end (outlet) and stack at one side of the boundary between the sample zone and the BGE, while the cations and neutral species move and exit the capillary at the injection end (inlet). Compared to FASS, this preconcentration method can provide much larger sample injection without any significant loss in separation efficiency, but it cannot be used for the simultaneous separation of anions and cations and, also, it is limited to analytes with low mobilities. Among the most frequently encountered methods for enhancing sensitivity in the analysis of drugs of abuse by the MEKC mode are the normal stacking and sweeping [48], especially combined with cation-selective exhaustive injection (CSEI), the so-called CSEI-sweeping method, which was first reported by Quirino and Terabe [49]. It results in more sensitive detection than sweeping and is sufficiently flexible to offer the potential to achieve an increase in the LOD of more than 100,000 fold, for positively charged analytes. At the beginning of the runs, the capillary is conditioned with a non-micellar BGE, followed by the injection of a high-conductivity buffer and, finally, the injection of a short water plug. Using electrokinetic injection at a positive polarity, the cationic analytes accumulate in a low-conductivity matrix or water. They enter the capillary through the water plug at high velocities and are then focused or stacked at the interface
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between the water zone and the high-conductivity buffer. Once an optimized injection time is determined, it is stopped and the micellar BGEs are replaced at both ends of the capillary. The voltage is then switched to negative polarity, thus permitting the entry of micelles from the cathodic vial into the capillary to sweep the analytes stacked and introduced to the narrow bands. Finally, the separation can be performed using MEKC in reversemigration mode. Compared to conventional off-line sample-preconcentration methods (LLE and SPE), the stacking methods are rather economical, sensitive, rapid, simple and reproducible.
3. Capillary electrophoresis with different detection systems 3.1. UV detection CE-UV coupling is the most widely used CE mode for the analysis of many drugs of abuse in biological samples, mainly urine. This type of detector is standard in commercial instruments, which can also be fitted with a diode-array detector for the simultaneous acquisition of spectra [50] However, the short internal diameter of the capillary (detection pathlength) limits the sensitivity of this detection system, and, consequently, LODs for UV-Vis absorption are usually not low enough for the quantification of drugs of abuse in biological samples. Thus, most of these applications require CE sensitivity to be enhanced by using more sensitive detection systems or by involving on-line or off-line sample-preconcentration methods, as discussed above. One of the first applications was by Wernly and Thormann in 1991 [51], who employed the MEKC mode for the qualitative analysis of different drugs of abuse and/or their metabolites. The peak identification was based on not only migration times, but also the on-line
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recorded UV spectra of the peaks. After SPE of 5 mL of urine, drug concentrations down to the ng/mL level were analyzable in this biological matrix. The same authors [52] showed that, using direct injection of urine, it was possible to achieve an LOD of about 20 lg/mL for the major metabolites of heroin and morphine-3-glucuronide using CZE and MEKC with UV detection. Lower LODs were obtainable when off-line preconcentration by SPE was employed. Highly specific analysis with short sample preparation with SPE and good recovery were obtained for a mixture of two opium alkaloids and two anesthetics in urine [53]. The group of McCord [54] developed and validated a method to screen urine for 19 different drugs of abuse, but the selectivity of the system was limited by the use of UV detection at 214 nm, a wavelength at which many endogenous compounds absorb. To avoid interferences from the matrix, an automated sequential injection SPE provided a preconcentration effect that gave very low LODs. UV detection was also employed at a very low wavelength (190 nm) with CZE and MEKC mode in the presence of SDS, enabling the analysis of morphine in human plasma at therapeutic concentrations [55]. A screening method for opiates, amphetamine and caffeine in urine and serum was reported by Hyo¨tyla¨inen et al. [56] by using MEKC-UV with an analysis time of only 2 min. Also, eight urine samples obtained from individuals undergoing methadone therapy were analyzed by CZE-UV without interference from coextracted drugs of abuse and/or their metabolites [57]. Another CE-UV method was proposed by Brown et al. [58] for the analysis of three common club drugs requiring minimal sample preparation and urine-sample volume with a run time of less than 7 min. Also, different substances related to heroin addiction and treatment were analyzed by CEUV with appropriate LODs [59]. Different amphetamines were analyzed in human whole-blood samples by CE-UV in less than 7 min, without the need of any
Figure 1. Electropherogram of (1) amphetamine, (2) methamphetamine, (3) 3,4-methylenedioxyamphetamine, (4) 3,4-methylenedioxyethyl amphetamine, (5) 3,4-methylenedioxymethamphetamine and (6) 2,5-dimethoxy-4-methyl-phenethylamine with DAD detection at 200 nm. CE conditions: capillary (50 cm x 50 lm internal diameter), temperature, 25C, running solution, 100 mM phosphate buffer (pH 2.5); voltage, 10 kV. With permission from ref. [60].
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derivatization procedure (see Fig. 1) [60], but needing a simple extraction method to obtain very good sensitivity. Coupling of CZE with UV detection was first used for the determination of illicit drugs in hair samples by Tagliaro et al. in 1993 [61]. The extraction procedure, preceded by an acidic incubation for 24 h, attained the LODs required for this type of sample (about 0.2 ng/mg). The same research group carried out a very convenient method with very good recoveries for routine analysis of cocaine in hair by employing CE-DAD [62]. As mentioned above, b-CD can be used to enhance selectivity in enantiomeric resolution being carried out by CE [18]. Qi et al. [63] used a multi-target inmunoaffinity column prior to CE separation with UV detection using b-CD as additive for the determination of morphine and related compounds in urine of heroin abusers obtaining very good reproducibility and precision. The method proposed by Liau et al. [64] also permits the chiral separation of methamphetamine, methacathionone, ephedrine and pseudoephedrine using b-CD in a CE-UV method. The determination of enantiomers in clandestine tablets and urine samples represents a good complementary method to GC-MS for use in forensic and clinical analysis. b-CD was also employed for the simultaneous separation of six drugs of abuse in urine and hair samples by CE-UV after a simple LLE extraction [65]. To increase the sensitivity required to carry out hair analysis, a FASS on-column preconcentration procedure was applied. The same chiral selector, added to a phosphate buffer, also offers excellent resolution and sensitivity for the analysis of human urine and hair samples from 6-acetylmorphine users [66]. A simple work-up procedure based on LLE permits very good recoveries. The simultaneous determination of morphine, codeine, 6-acetylmorphine and normorphine was
also carried out using CE-UV with b-CD in the BGE. The application of electrokinetic injection with FASS resulted in low LODs (40 ng/mL) [67]. To obtain a 1000-fold sensitivity enhancement, the FASS on-column sample-preconcentration procedure was applied to the determination of opioids in urine [68]. The same detection system was used in CE for the analysis of four amphetamines applying electrokinetic injection with FASS in urine samples [69]. Also, Taylor et al. [70] provided an excellent quantitative determination of opiates using a BGE of disodium hydrogen phosphate at neutral pH, obtaining very good results when carrying out the methodology in the same biological matrix. The FASS procedure was also applied to the analysis of hair samples to increase the sensitivity required for this type of biological specimen. The simultaneous analysis of morphine, cocaine and 3,4-methylenedioxymethamphetamine was carried out with CE-UV [71]. Also, the FASS procedure was used for the determination of different major opiates in hair samples, employing acidic BGE in the presence of organic solvent [72]. Four amphetamines in spiked hair samples were also analyzed by CZE using this type of preconcentration procedure that increased detection sensitivity by up to about 1000-fold [73]. The CSEI-sweeping sample-preconcentration procedure, usually employed in MEKC mode, was used for the determination of heroin, morphine, codeine and 6-monoacetylmorphine in urine samples [74]. The analysis was carried out in less than 10 min and only a simple LLE extraction was needed to get rid of salts. Lin et al. [75] developed another CSEI-sweep MEKC method to determine morphine, codeine, normorphine, morphine-3-glucoronide and morphine-6-glucoronide also in urine samples, obtaining a sensitivity increase of 2500-fold with respect to the CZE mode. The same on-
Figure 2. Separation of three amine drugs by using CSEI-sweeping. (1) 3,4-methylenedioxymethamphetamine, (2) methamphetamine (3) amphetamine and (IS) benzylamine at a concentration level of 5 ng/mL. With permission from ref. [76].
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Figure 3. Electrochromatopherograms resulting from the analysis of (a) blank urine sample, (b) urine sample spiked with a standard mixture of: (1) amphetamine, (2) methamphetamine, (3) 3,4-methylenedioxyamphetamine, (4) 3,4-methylenedioxymethamphetamine, (5) 3,4-methylenedioxyethylamphetamine, (6) heroin, (7) codeine, (8) 6-monoacetylmorphine, (9) cocaine and (10) morphine. The sample was electrokinetically injected (7 kV x 10 s); mobile phase, 20 mM phosphate (pH 2.5)/acetonitrile (80/20, v/v). With permission from ref. [21].
column preconcentration procedure was developed for the determination of cocaine and its metabolites in urine [76]. The BGE was a mixture of phosphoric acid, SDS, 2-propanol and tetrahydrofuran, and, using an electrokinetic sample injection, led to 1000-fold reductions in LODs. The applicability of this preconcentration procedure in the analysis of forensic samples was also demonstrated in the determination of methamphetamine, ketamine, codeine and morphine in hair samples employing MEKCUV mode [75]. As seen in Fig. 2, the combination of CSEI with the MEKC mode also lowered by down to 1000-fold the LOD of three amphetamines (3,4-methylenedioxymethamphetamine, methamphetamine, amphetamine and benzylamine) in hair samples using a BGE in presence of SDS and an organic solvent [77]. The CSEI-sweeping sample-preconcentration procedure was also employed for separation and determination of basic compounds of forensic interest in human urine. Fig. 3 shows the electropherograms corresponding to a blank urine sample and spiked samples, treated following the proposed procedure [50]. A very interesting methodology employing a particular type of stacking called low temperature bath (LTB) stacking, coupled with the NACE mode, was used for the analysis of 3,4-methylenedioxymethamphetamine in illicit drug and urine samples [78]. A portion of the capillary was immersed in a low-temperature bath, which served as a ‘‘pseudo-low-conductivity zone’’ and, as a result, a large volume of sample injection could be achieved, lowering the LODs dramatically.
3.2. Fluorescence detection FL-HPLC detection has been widely used in many fields of analytical chemistry with some examples in the field of forensic analysis [23,79,80] because of the sensitivity and the selectivity that this detection system can offer. However, on-column FL detection has sensitivity limitations, due to the difficulties in focusing enough excitation energy into the capillary and efficiently collecting the radiation emitted [23]. In such cases or in those where the analytes do not exhibit strong enough native FL, they can be derivatized with a proper derivatization agent, but the advantage of detecting native FL is that no precolumn-derivatization step is necessary, simplifying the chemical work-up and reducing the time of total analysis. The lack of significant native FL of most of the molecules can be an important advantage of FL detection if selectivity is required. Another way to enhance sensitivity and selectivity greatly is the combination of CE with a LIF detector, requiring the appropriate laser sources. The use of FL/LIF detection in forensic analysis is mainly restricted to urine samples. Methods for separation and determination of normorphine, morphine, 6-acetylmorphine and codeine in biological fluids using CE and LIF detection were described by Alnajjar et al. [81]. When the assay was based on native FL, LODs in the range of 200 ng/ml were achieved. To enhance sensitivity further, a two-step precolumn-derivatization procedure using 1-chloroethyl chloroformate and fluorescein isothiocyanate (FITC) as the reagents was employed before LIF detection, leading to LODs at the 50–100 pg/mL level.
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Phenethylamine designer drugs were determined in urine samples by MEKC-LIF based on their native FL and using LLE and sweeping preconcentration to improve the LODs [82]. Using FITC as derivatization agent before a CELIF method, six illicit drugs (ephedrine, pseudoephedrine, amphetamine, methampetamine, 3,4-methylendioxyamphetamine and 3,4-methylendioxymethylamphetamine) were simultaneously determined with an LOD of 200 pg/mL [83]. 3.3. Mass-spectrometry detection To overcome the drawbacks of UV and FL detection modes discussed above, CE coupled with MS was recently established as a powerful method for forensic screening [84,85]. This technique, with its excellent detection sensitivity and with the special characteristic of providing structural information on the analytes, has become complementary to HPLC-MS [86,87] and is especially useful for the analysis of matrices (e.g., hair) with low drug content. However, due to the high price and limited commercial availability of CE-MS instruments, this technique is not yet in general use in forensic analysis. In CE-MS, the soft ionization techniques are also in use in HPLC-MS. Currently, electrospray ionization (ESI) is the most popular interface between CE and MS, because it facilitates the transfer of analytes from the liquid phase of the CE to the gas phase of the MS. Atmospheric pressure electron-impact (APEI) ionization also gave very good sensitivity and selectivity for a wide range of analytes of clinical and forensic significance [88]. Using
an ESI interface, da Costa et al. determined cocaine and its major metabolites in human urine [89]. The coupling of CE-ESI-MS, preceded by simple SPE, has also enabled the determination of different derivatives of 2,5-dimethylamphetamine [90]. With the same interface, another method was proposed to screen and to quantify four thiophenethylamine designer drugs in human plasma after a simple LLE for sample clean-up [91]. 3,4-methylenedioxy-methamphetamine and methadone were determined using protein precipitation prior to the hydrodynamic injection of the samples [92]. A method has been proposed by Gottardo et al. for the determination of illicit drugs in blood samples after LLE and FASS on-column preconcentration using CZE with ESI and time-of-flight (TOF)-MS [93]. 3,4-methylenedioxymethamphetamine and three of its main metabolites were also analyzed in plasma and urine samples after administration of ecstasy [94]. Ion-trap (IT) MS is also a useful tool for forensic analysis. Confirmation testing of urinary amphetamines and designer drugs by CE/IT-APEI-MS gave very good results [95]. Wey and Thormann [68] also applied a CE-MS with FASS to the determination of opioids in urine samples, resulting in a 1000-fold sensitivity enhancement with respect to UV detection. The same authors [96] used CEMS with APEI for the analysis of morphine and related opioids in the same biological specimen with hydrody-
Table 1. Analytical methodologies for drugs of abuse in blood samples Analytes Morphine
Morphine, heroin, codeine, amphetamine, caffeine Amphetamine, methamphetamine, 3,4methylenedioxyamphetamine, 3,4methylenedioxyethylamphetamine, 3,4methylenedioxymethamphetamine, 2,5dimethoxy-4-methyl-phenethylamine Thiophenethylamine designer drugs (2CT-series) Methamphetamine, 3,4methylenedioxyamphetamine, 3,4methylenedioxyethylamphetamine, 3,4methylenedioxymethamphetamine, methadone, cocaine, benzoylecgonine, morphine, codeine and, 6acetylmorphine) 3,4-methylenedioxymethamphetamine, 3,4-methylelenedioxyamphetamine, 4hydroxy-3-methoxymethamphetamine and 4-hydroxy-3-methoxyamphetamine Ecstasy and methadone
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Experimental conditions
Mode of detection
LOD
Reference
BGE of disodium tetraborate decahydrate (pH 10.5) and SDS (50 mM) 50 mM glycine and 50 mM SDS (pH 10.5) BGE of 100 mM phosphate buffer (pH 2.5)
CZE-UV (190 nm) MEKC-UV
50 ng/mL
[55]
MEKC-UV
0.34–1.20 lg/mL
[56]
CZE-UV (200 nm)
10–30 ng/mL
[60]
LLE
CE-ESI-MS
11.3–23.0 ng/mL
[91]
LLE FASS, ammonium formate (25 mM) (pH 9.5)
CE-ESI-TOF-MS
2–10 ng/mL
[93]
CE-ESI-TOF-MS
2–10 ng/mL
[94]
CE-MS
0–175 ng/mL
[92]
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Table 2. Analytical methodologies for drugs of abuse in urine samples Analytes Benzoylecgonine, morphine, heroin, 6acetylmorphine, methamphetamine, codeine, amphetamine, cocaine, methylephedrine, methaqualone, benzodiazepines Morphine-3-glucoronide Lidocaine, bupivacaine, noscapine, papaverine 19 drugs of abuse Methadone Methamphetamine, 3,4methylenedioxyamphetamine, ketamine Morphine, codeine, methadone, 2-ethylidene1,5-dimethyl-3,3-diphenylpyrrolidine Morphine, codeine, acetylcodeine, 6acetylmorphine, morphine-3-glucoronide 3,4-methylenedioxymethamphetamine, 3,4methylenedioxyamphetamine, ephedrine, amphetamine, methamphetamine Methamphetamine, methacathionone, ephedrine and pseudoephedrine Opioids Amphetamine, methamphetamine, 3,4methylenedioxyamphetamne, 3,4,methylenedioxymethamphetamine Pholcodine, 6-monoacetylmorphine, morphine, heroin, codeine, dihydrocodeine Heroin, morphine, codeine, 6-acetylmorphine Morphine, codeine, normorphine, morphine-3glucoronide, morphine-6-glucuronide Cocaine, benzoylecgonine, norcocaine, cocaethylene Morphine, heroin, codeine, amphetamine, caffeine Amphetamine, methamphetamine, 3,4methylenedioxyamphetamine, 3,4methylenedioxymethamphetamine, cocaine, codeine, heroin, morphine, 6-acetylmorphine 3,4-methylenedioxymethamphetamine 2C-T-2, 2C-T-7, 2c-c. 2c-B, « c-I Normorphine, 6-acetylmorphine, codeine, morphine Pseudoephedrine, ephedrine, amphetamine, methamphetamine, 3,4methylenedioxyamphetamine, 3,4methylenedioxymethamphetamine Normorphine, codeine, 6-acetylmorphine Cocaine, benzoylecgonine, cocaethylene, anhydroecgonine, anhydroecgonine methyl ester, ecgonine methyl ester
2,5-dimethylamphetamine and derivatives
Experimental conditions
Mode of detection
LOD
References
SPE Borate/phosphate buffer (75 mM) with SDS
MEKC-UV
100 ng/mL
[51]
Sample extraction with C8 cartridges SPE BGE with b-CD and organic. SPE Extraction with disposable cartridges
CZE-UV MEKC-UV CZE-UV CE-UV
20 lg/mL 1 lg/mL 300 ng/mL 5–30 ng/mL
[52]
CZE-UV
20 ng/mL
[57]
CE-UV
25–300 lg/mL
[58]
[53] [54]
CE-UV Inmmunoaffinity column and b-CD b-CD and LLE
[59]
CE-UV
10–20 ng/mL
[63]
CE-UV(200 nm) CE-DAD (190–240 nm)
500 ng/mL
[65]
b-CD
CE-UV
FASS
CZE-UV
25–34 lg/mL
[68] [69]
FASS and SPE
CZE-UV
4–9 ng/mL
[70]
CSEI-sweeping and LLE CSEI-sweeping
MEKC-UV MEKC-UV
10 ng/mL 10–35 ng/mL
[74] [75]
CSEI-sweeping
MEKC-UV
–
[76]
50 mM glycine and 50 mM sodium dodecyl sulfate (pH 10.5) SPE on strong cation exchange cartridges
MEKC-UV
0.34–1.20 lg/mL
[56]
CEC-UV
5–12 ng/mL
[21]
Low temperature bath stacking Sweeping and FITC derivatization ACE-CI and FITC
NACE-UV
10
CE-FL MEKC-LIF CE-FL CE-LIF CE-LIF
10 7–10
200 ng/mL 50–100 pg/mL 200 pg/mL
[81]
CE-FL CE-LIF CE-ESI-MS
200 ng/mL 50–100 pg/mL 100–250 ng/mL
[67]
CE-ESI-MS
10–1000 ng/mL [90] (continued on next page)
Derivatization with FITC
Formic acid (1 M) as electrolyte and formic acid (0.05 M) in methanol:water as coaxial sheath liquid SPE
[64]
6
M–5.0 x 10 8
M
9
M
[78] [82]
[83]
[89]
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Table 2. (continued) Analytes 3,4-methylenedioxymethamphetamine, 3,4methylelenedioxyamphetamine, 4-hydroxy-3methoxymethamphetamine, 4-hydroxy-3methoxyamphetamine Amphetamine, methamphetamine, 3,4methylenedioxymethamphetamine, 3,4methyelenedioxyamphetamine Opioids Morphine and related opioids
Experimental conditions
CE-ESI-TOF-MS
BGE of ammonium acetate and acetic acid (pH 4.6) FASS Hydrodynamic sample injection
namic sample injection. The LODs were in the ng/mL level. Zhang et al. recently reported a CE-MS method based on a charged polymer-protected gold nanoparticlecoated capillary for the analysis of heroin and its basic impurities [97]. Although some limitations regarding the selection of CE-electrolyte composition and the concentration sensitivity of the CE-MS coupling have not yet been completely overcome, several applications have been published in the field of drugs-of-abuse analysis in hair [98,99]. Gottardo et al. [98] developed a simple, rapid CZE-IT-MS method for the sensitive, quantitative determination of different drugs of abuse and their metabolites in human hair, applying FASS preconcentration to lower the LODs to the severe cut-offs adopted in this type of analysis (i.e. 0.1 ng/mg) for both clinical and forensic purposes. When coupling CE-ESI-MS and CE-TOF-MS with a simple LLE, off-line preconcentration, the determinations of different drugs in the same matrix at the low concentrations required were also obtained [99]. Another CZE-ESI/TOF-MS method was developed and validated for the identification and determination of numerous drugs of abuse in blood and hair samples [93]. A confirmatory assay based on SPE extraction followed by CE-ESI-MS has been employed for the simultaneous analysis of enantiomers of methamphetamine, amphetamine, 2,5-dimethylamphetamine, ephedrine, norephedrine and methylephedrine hydrochloride in urine samples [100]. The same ionization process was employed for the analysis of human urine [101] after the administration of codeine and codeine6-glucoronide.
4. Applications to biological samples Typically, the biological samples subjected to drug analysis for forensic toxicology purposes are widely different and include almost any tissue and organ, but blood, urine, saliva, hair and nails are most important for detecting drugs of abuse [102]. In order to limit the
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Mode of detection
LOD 2–10 ng/mL
CE-API-MS CE-IT-MS CE-MS CE-MS CE-APEI-MS
References [94]
[95]
100–200 ng/mL
[68] [96]
workload due to the huge increase of casework occurring in recent decades, there is a tendency to limit as much as possible the specimens to be analyzed to those having a fundamental value for the purpose of the investigation [103]. In accordance with this tendency, we have focused our attention in this review to blood, urine and hair, which represent the majority of specimens analyzed for drugs of abuse in most laboratories. 4.1. Analysis of blood samples Plasma and serum are typically used for emergency testing in forensics and toxicology, and in therapeutic monitoring, as they are suitable as samples to determine the short-term use of drugs. Drawbacks are that blood is an invasive sample, difficult to handle and reflects the patientÕs dosages for some hours only. Some examples are shown in Table 1. As seen in Table 1, UV and MS are the two detection systems employed, and, since the sensitivity required in this biological sample is higher than in urine, off-line preconcentration procedures, such as LLE, are applied. 4.2. Analysis of urine samples Urine is the most commonly used biological sample for the analysis of drugs of abuse (e.g., amphetamines and opiates) in forensic laboratories. It is generally used for the detection of these compounds in schools and workplaces, because it is obtainable non-invasively, as compared to blood sampling. The main limitation in its use is that it provides direct evidence only for short-term use of illicit drugs because these remain in urine no longer than for 10 days after use [104]. Also, urine analysis cannot distinguish between habitual and first-time users. Nevertheless, as seen in Table 2, most of the applications of CE methodologies found in the literature are carried out with urine. UV detection generally used does not usually offer the sensitivity required for this type of analysis. For this reason, various off-line and on-line preconcentration procedures are usually employed, alone or in combination (e.g., LLE or SPE with FASS or CSEI-sweeping), resulting in LODs at the ng/mL level.
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Table 3. Analytical methodologies for drugs of abuse in hair samples Analytes Cocaine, morphine
Cocaine Methamphetamine, methacathionone, ephedrine, pseudoephedrine Ephedrine, amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methyelenedioxyamphetamine, 3,4-methylenedioxyethylamphetamine Morphine, cocaine, 3,4-methylenedioxymethamphetamine Morphine, codeine, 6-monoacetylmorphine, acetylcodeine, heroin Amphetamine, methamphetamine, 3,4-methyelenedioxyamphetamine, 3,4-methylenedioxymethamphetamine Methamphetamine, ketamine, codeine, morphine 3,4-methyelenedioxyamphetamine, methamphetamine, amphetamine, benzylamine 6-monoacetylmorphine, morphine, amphetamine, methamphetamine, 3,4-methyelenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, benzoylecgonine, ephedrine, cocaine Amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethaamphetamine ephedrine, cocaine, morphine, codeine, 6-acetylmorphine, benzoylecgonine Methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxyethylamphetamine, 3,4-methylenedioxymethaamphetamine methadone, cocaine, benzoylecgonine, morphine, codeine, 6-acetylmorphine
Experimental conditions
Mode of detection
Acidic incubation overnight in 0.25 M HCl (45C) and extraction with Toxi-tubes 50 cm x 75 lm capillary
LOD
Reference
CZE-UV (214 and 238 nm)
0.15 ng/mg
[61]
CE-DAD (200 to 400 nm)
0.15–0.35 ng/ mg 300 ng/mL
[62] [66]
150 mM phosphate buffer (pH 2.5) + 15 mM b-CD FASS b-CD (15 mM)
CE-UV (200 nm) CE-UV (200 nm) or multiwavelength (190– 400 nm)
0.2 lg/mL
[65]
FASS
CE-UV (200 nm) or multiwavelength (190– 400 nm) CE-UV
2–8 ng/mL
[71]
0.75 ng/mL to 150 pg/mL
[72]
FASS, BGE (sodium phosphate + 40% ethylene glycol (pH 2.5) FASS, BGE (phosphate buffer, 100 mM + 40% ethylene glycol (pH 2.5) CSEI-sweeping
CZE-UV
0.06 lg/mL
[73]
MEKC-UV
50–100 pg/mL
[75]
CSEI-sweeping
MEKC-UV
0.01 lg/mL to 50 pg/mL
[77]
FASS
CE-IT-MS
0.1 ng/mg
[98]
LLE
CE-ESI-MS CE-TOF-MS
0.1 ng/mg
[99]
FASS LLE
CZE-ESI-MS CZE-TOF-MS
0.1 ng/mg
[93]
Using FL (especially LIF) detection, LODs can be decreased to the pg/mL level. But because most of the drugs of abuse under study do not present native FL, a previous derivatization step with a derivatization reagent, usually FITC, is often required, complicating the analysis methodology. Due to its advantages discussed in sub-section 3.3, CE-MS is becoming the most desirable method in forensic laboratories. 4.3. Analysis of hair samples Undoubtedly, urine and blood are the routine samples of choice for drug analysis, but, beside them, hair is being recognized as the third fundamental biological specimen for drug testing [105]. Although more than 450 papers dealing with hair analysis for drugs have been published
since 1954, most of them have appeared in this decade [106]. One of the reasons for this increase is that hair can provide a historical account of drug intake or exposure, whereas fluid matrices (e.g., the previously cited urine or blood) can give information about only fairly recent drug use [107]. Hair analysis is currently applied in workplace drug testing, post-mortem toxicology, doping control and certification of physical fitness to drive vehicles. Also, it has been used to investigate ancient peoplesÕ lives (e.g., diet and cause of death) in archaeological studies [108], but its maximum potential is in forensic toxicological and drugs of abuse studies. The use of hair in forensic toxicology was introduced in the late 1970s by Baumgartner et. al. [109], and,
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from the analytical toxicology point of view, it poses specific problems, especially sample collection, preparation and extraction. Nevertheless, it has demonstrated many advantages compared with blood and urine. As a sample, it is a obtainable non-invasively, it is easy to preserve and it is the only one that provides information about the quantity and historic pattern of drug use, having a retrospective period of months or years, saving drugs and their metabolites unchanged. The reason is that drugs enter the hair stalk at the hair root together with the nutrients from the capillary blood and remain embedded in the of hair matrix for all the life of this structure (from months to years). Since the rate of growth of hair is about 1 cm per month, each cm of hair keeps the record of about 1 month of use or exposure of the individual to drugs. This type of information is not available from any other specimen. Only a limited number of papers have described methodologies for hair analysis based on CE (see Table 3). The contribution of the group of Tagliaro from the University of Verona is remarkable [61,62,65, 66,71]. The detection system most frequently employed, at present, is UV, which does not give the required sensitivity in this type of biological matrix. For this reason, it is usually necessary to apply a sample-preconcentration procedure, FASS being employed most often. However, the most recently published methods for the analysis of drugs of abuse in hair employed MS as the detection system [10,93,99], so CE-MS will be the most important technique for this type of analysis in forensic laboratories in the near future. References [1] [2] [3] [4]
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