Chapter 11 Forensic screening by gas chromatography

M.J. Bogusz (Ed.). Forensic Science Handbook of Analytical Separations, Vol. 6 r 2008 Elsevier B.V. All rights reserved

403

CHAPTER 11

Forensic screening by gas chromatography Ilkka Ojanpera¨ and Ilpo Rasanen Department of Forensic Medicine, P.O. Box 40, FI-00014 University of Helsinki, Finland

11.1 INTRODUCTION Discovered already in 1952 by A.T. James and A.J.P. Martin [1], gas chromatography (GC) was introduced to forensic toxicological drug analysis during the 1960s [2], but it only became popular in the 1970s. The technique offered a long-awaited solution for quantitative analysis of drugs in blood, replacing earlier techniques in this area, such as UV spectrometry and thin-layer chromatography (TLC). In 1967, M.W. Anders and G.J. Mannering wrote in Progress in Chemical Toxicology: ‘‘The use of gas chromatography in chemical toxicology is in its infancy. Whereas its greatest usefulness will ultimately be realized in the screening for specimens in the ‘general unknown’ toxicological analysis, it is currently used largely in special procedures designed for the analysis of a single agent or class of agents y’’ [3]. Indeed, as packed columns offered only limited separation efficiency, their use was first limited to quantitative target analysis of substances previously identified by TLC in the urine or liver. Along with the development of column oven temperature programming and better and more reproducible packed columns, comprehensive drug screening methods emerged [4]. This was followed by the establishment of comprehensive GC retention index (RI) libraries for the most common stationary phases, allowing cross-referencing with the retention data generated by other laboratories [5]. However, it was only the capillary column, and especially the fused silica column technology [6], that started the era of modern GC screening in the early 1980s. The most active years of developing and publishing screening methods by capillary GC were in the 1980s and early 1990s. That GC gradually became a mature technology and was more often connected with mass spectrometry (GC-MS) lowered the frequency of published non-MS methods but did not change the importance of GC as a workhorse in the laboratory. Instead, progress in electronics and pneumatics resulted

References pp. 423–424

404

Chapter 11

in improved precision of retention parameters through retention time locking (RTL) [7], and this benefit has further strengthened the role of GC in toxicological screening. This chapter concentrates on the screening of toxicological samples for drugs by capillary GC using techniques other than MS detection. The key published papers, listed in Tables 11.1 and 11.2, are divided according to the use of a single column or two parallel columns, respectively. These studies describe general methods for comprehensive drug screening or demonstrate a potential for such an application; narrow target analyses are not included. Screenings in pesticide and doping control are discussed in their respective chapters. Other screening techniques based on GC-MS, high-performance liquid chromatography (LC) and LC-MS are treated elsewhere in the book. 11.2 SAMPLE PREPARATION Most GC screening methods have been developed mainly for blood (plasma, serum) samples. This is logical because GC by nature is at its best with non-polar analytes, such as the parent compounds of psychopharmaceuticals, which are present in blood at appropriate concentration levels. Urine, on the other hand, contains more polar metabolites that need derivatization to be detected by GC. In comprehensive screening procedures aiming for high throughput, the sample preparation should be kept effortless. Although derivatization improves the limit of detection (LOD) and limit of quantification (LOQ) of compounds possessing an – OH or –NH group, particularly acids, phenols, imides and secondary and primary amines and amides, it has only occasionally been used in comprehensive screening procedures. Instead, simple liquid–liquid solvent extraction (LLE) with ethyl or butyl acetate, butyl chloride or methyl tert-butyl ether, is commonly carried out at pH 3–7 and pH 9–11 to fractionate acidic/neutral and basic drugs, respectively. Extraction with a fairly non-polar high-boiling solvent, such as butyl acetate, produces clean extracts, with the upper layer easily removable for direct injection into GC. Notably, LLE on a small scale, starting from p1 ml of blood and p0.5 ml of organic solvent, is both practical and economical. However, putrefied blood samples require a clean-up stage to prevent analytical columns from deteriorating. Solid-phase extraction (SPE) has been used more rarely in connection with GC screening, as it is a more expensive, labour-intensive and vulnerable technique. However, an SPE method even for whole blood has been developed by Chen et al. [20]. Flanagan et al. [42] have recently discussed in detail various micro-extraction techniques in analytical toxicology, including micro-LLE, salting-out, extractive derivatization, protein precipitation, solid-phase micro-extraction and liquid-phase micro-extraction. 11.3 SEPARATION 11.3.1 General Fused silica capillary columns have been used since the early 1980s for the screening of drugs and poisons in GC. Bonded polysiloxanes are the most popular stationary

Reference Eklund et al.b(Sweden 1983) [8] Dunphy and Pandya (USA 1983) [9] Anderson and Stafford (USA 1983) [10] Ehresman et al. (USA 1985) [11] Taylor et al. (USA 1986) [12]

Soo et al. (USA 1986) [13] Sharp (Canada 1986) [14] Anderson and Fuller (USA 1987) [15] Sharp (Canada 1987) [16] Caldwell and Challenger (UK 1989) [17]

Number of Drugs

Sample

Extraction

Limit of Detection (LOD) or Quantification (LOQ)

Column

a

Detector

Identification Method

80 basic

Liver

LLE, butyl acetate

SE-52

NPD

RRT

18 acidic and neutral

Serum

LLE, dichloromethane

DB-5

NPD

RRT

175 basic

Blood or urine

LLE, butyl chloride

SE-30

FID

RI

56 acidic and basic

Body fluids

LLE, dichloro-methane

25 basic

Urine

LLE, hexane: isoamyl alcohol 95:5

11 hypnotic-sedative

Serum

170 basic

Retention Index Standards

alkanes (external)

Confirmation GC-MS or TLC if necessary TLC or immunoassay GC-MS

LOD generallyp1 mg/l LODX0.05 mg/l

SE-54

FID

RRT

GC-MS

DB-1701

NPD

RRT

LOD 0.5 mg/l

HP-5

NPD

RRT

Blood

LLE, dichloro-methane (Toxi-Tube B) LLE, butyl chloride

confirmation method for TLC and immunoassay GC-MS

LODo0.2 mg/l

HP-1, 0.53 mm

NPD

RT

52 acidic and neutral

Blood

LLE (Chem Elut)

LOD 1–20 mg/l

HP-1, 0.53 mm

FID

RI

60 acidic and neutral

Blood

LLE, ethyl acetate

LODo10 mg/l

DB-1

FID

RI

300 basic

Urine

LLE, butyl acetate

LODo0.25 mg/l

HP-5

NPD

RRT

119 basic

Blood

LLE, butyl acetate

LOD 0.1–0.2 mg/l

HP-17

NPD

RRT

alkanes (external) alkanes (external)

Forensic screening by gas chromatography

References pp. 423–424

TABLE 11.1 SINGLE-COLUMN CAPILLARY GAS CHROMATOGRAPHIC METHODS FOR DRUG SCREENING

GC-MS GC-MS

405

used with TLC and immunoassay GC-MS

406

TABLE 11.1 CONTINUED

Reference Cox et al. (USA 1989) [18] Drummer et al. (Australia 1994) [19] Chen et al. (The Netherlands 1994) [20] Lillsunde et al. Finland 1996) [21]

Number of Drugs

114 neutral and basic

Sample

Extraction

Limit of Detection (LOD) or Quantification (LOQ)

a

Column

Detector

Identification Method

Blood or plasma Plasma, urine or blood

LLE, butyl chloride

LOD 0.02–0.5 mg/l

BP-5

NPD

RRT

SPE (Bond Elut Certify)

LOD 0.1–0.2 mg/l

HP-1

NPD

RRT

10 acidic

Blood

LOD 0.25–1 mg/l

HP-5

NPD

RRT

Tokunaga et al. (Japan 1996) [22]

12 basic

Plasma

LLE, toluene:ethyl acetate 8:2, methylation (PHMAH) LLE, methyl t-butyl ether SPE (Sep-Pak C18)

CBP-1, 0.53 mm

NPD

RT

Soriano et al. (Spain 1996) [23] Sanchez de la Torre et al. b(Spain 2005) [24]

34 basic and neutral

Urine

LOD 0.01–0.2 mg/l LOD 0.005– 0.05 mg/l LOD 0.1–0.8 mg/l

HP-5

NPD

RT

8 psychiatric

Blood

HP-1

NPD

RRT

18 basic

LLE, methyl t-butyl ether, (PrepStation) SPE (Bond Elut Certify)

LOD 0.037– 0.156 mg/l LOQ 0.12–0.52 mg/l

Retention Index Standards

Confirmation

GC-MS or HPLC

GC-MS

Abbrev.: LLE: liquid-liquid extraction; SPE: solid-phase extraction; PHMAH, phenyltrimethylammonium hydroxide; a

Column internal diameter is 0.2–0.32 mm, if not otherwise stated. NPD, nitrogen-phosphorus specific detector; FID: flame-ionization detector; RRT, relative retention time; RI, retention index; RT: absolute retention time; GC-MS, gas chromatography – mass spectrometry; TLC, thin-layer chromatography, HPLC, high-performance liquid chromatography.

b

Includes validation data for quantification.

Chapter 11

Limit of Detection (LOD) or Quantification (LOQ)

Detectors

Identification method

SE-54 OV-101

2 x NPD

RRT

LLE, ethyl acetate

SE-54 OV-215

NPD, FID RRT

Ultra 1 Ultra 2

2 x FID

RI

Blood

LLE, dichloro methane:isopropanol 95:5 (ClinElut) LLE, toluene

LOQ generally 0.2 mg/l

DB-1 DB-1701 nonsimultaneous

2 x NPD

RRT

47 basic

Serum, body fluids

LLE, hexane:isoamyl alcohol 98:2

LOD 0.01–0.2 mg/l

BP-1 DB-1701

2 x NPD

RRT

110 basic

Blood

LLE, butyl chloride

LOD 0.1 mg/l

Ultra 1 HP-17

2 x NPD

RRT

Tissues

2
SPE (Chem Elut)

CP Sil 8 CP Sil 19 CB

2 x NPD

RRT

Plasma

LLE, diethyl ether

LOD generally 0.1 mg/l

Ultra 1 CP Sil 19 CB

2 x NPD

RRT

LLE, hexane:diethyl ether 1:1

Therapeutic concentrations

DB-1 DB-17

2 x NPD

RI

LLE, hexane: dichloromethane 7:3

LOD 0.01–0.05 mg/l

2
SE-54

ECD, NPD

RT

Number of Drugs

Hime and Bednarczyk (USA 1982) [25] Alm et al. (Sweden 1983) [26] Newton and Foery (USA 1984) [27] Koves and Wells b (Canada 1985) [28] Fretthold et al. (USA 1986) [29] Watts and Simonick (USA 1986) [30] Cordonnier et al.b (Belgium 1987) [31] Turcant et al. (France 1988) [32] Manca et al. (Canada 1989) [33] Lillsunde and Seppa¨la¨b (Finland 1990) [34]

48 basic

Urine

LLE, butyl chloride:isoamyl alcohol 99:1

45

Illicit drug samples

33 acidic and neutral

Plasma

102 basic

200 neutral and basic 172 neutral and basic

Sample

Plasma, urine or gastric 21 benzodiazepines Plasma or blood

Extraction

LOD generally 0.05 mg/l

Columns

Retention index standards

Reporting

Confirmation other methods

software

alkanes (external)

TLC and UV

software

GC-MS

GC-MS

software

drugs (external)

APL *Plus 5.5 Software

407

Reference

a

Forensic screening by gas chromatography

References pp. 423–424

TABLE 11.2 DUAL-COLUMN CAPILLARY GAS CHROMATOGRAPHIC METHODS FOR DRUG SCREENING

408

TABLE 11.2 CONTINUED

Reference

Number of Drugs

Phillips et al. (USA 1990) [35] Ojanpera¨ et al.b (Finland 1991) [36]

200 neutral and basic

Blood or tissues

LLE, butyl chloride

LOD 0.01–0.3 mg/l

31 acidic and neutral

Blood

LLE, ethyl acetate

Therapeutic concentrations

alkylbis(trifluoromethyl) phosphine sulfides (internal) LLE, diethyl ether, silylation (MTBSTFA)

Micman 4.03 software

Kim et al. (South Korea 1991,1993) [37,38] Coudore et al.b (France 1993) [39] Lillsunde et al.b (Finland 1996) [21] Rasanen et al.b (Finland 2000) [40] WorkStation 3.0 software Rasanen et al.b (Finland 2003) [41]

Sample

Extraction

Limit of Detection (LOD) or Quantification (LOQ)

Detectors

Identification method

DB-1 DB-5 nonsimultaneous

FID

RI

NB-54 NB-1701

2
NPD

RI

DB-5 DB-17

2
FID

RI

Columnsa

26 acidic

Serum

9 barbiturates

plasma

LLE, chloroform

LOD 0.5 mg/l

SPB-1 SPB-20

NPD, FID RRT

40 basic

Blood

LLE, dichloromethane: toluene 1:9, acylation (HFBA) LLE, ethyl acetate, silylation (MTBSTFA)

LOD 0.01–0.1 mg/l

2
HP-5

NPD, ECD

RI

LOD 0.003–0.05 mg/l DB-5 DB-17 LOQ 0.0060.075 mg/l

2
ECD

LLE, butyl acetate

LOQ 0.02–5 mg/l

2
NPD

26 benzodiazepines Blood

124 basic

Blood

HP-5 DB-17

Retention index standards

Reporting

alkanes (external)

Confirmation GC-MS

alkanes (internal)

software

GC-MS

drugs (internal)

SC-ChromBooster software

GC-MS

RI

benzodiazepine RI standards

SC-

RRT with RTL

SC-ChromBooster software

a

column internal diameter is 0.2–0.32 mm, if not otherwise stated. NPD, nitrogen-phosphorus specific detector; FID, flame ionization detector; ECD, electron capture detector. RRT, relative retention time; RI, retention index; RT, absolute retention time; RTL, retention time locking. TLC, thin-layer chromatography; GC-MS, gas chromatography – mass spectrometry.

b

Includes validation data for quantification.

Chapter 11

LLE, liquid-liquid extraction; SPE, solid-phase extraction; MTBSTFA, N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide; HFBA, heptafluorobutyric anhydride;

Forensic screening by gas chromatography

409

phases due to such favourable properties as high thermal stability, good diffusivity and suitable solvation characteristics (solvent strength or polarity and selectivity). The use of more polar polyethyleneglycol phases is limited by their maximum operating temperature of 250–2801C. According to Table 11.1, the most popular stationary phases used in single column methods were non-polar 100% dimethylpolysiloxane (SE-30, HP-1, DB-1, etc.) and slightly polar 5% diphenylmethyl-polysiloxane (SE-52, HP-5, DB-5, etc.) or 1% vinyl 5% phenylmethyl-polysiloxane (SE-54), which possess the best thermal stability and a low column bleed. In only two methods were the intermediately polar columns 14% cyanopropylphenylmethylpolysiloxane (DB-1701) [12] and 50% phenylmethylpolysiloxane (HP-17) [18] used.

11.3.2 Dual-column approach The use of dual capillary column GC was first described by Phillips et al. [43] for analysis of essential oils, and the concept was soon applied to drug screening [25]. According to Table 11.2, in dual-column methods, the stationary phase combinations were non-polar/slightly polar, non-polar/intermediately polar or slightly polar/ intermediately polar. Two methods had two similar slightly polar columns and two different detectors [21,34]. The use of two parallel capillary columns of different polarity makes GC even more powerful than using a single column, judging from the relatively high number of reports that utilize this approach in drug screening. However, most papers do not present well-founded arguments to support the dual-column concept. It has also been stated that a second phase in addition to dimethylpolysiloxane would be of limited use because the high degree of correlation would give little or no extra information in general screening procedures [44]. Manca et al. [33] evaluated their dual-column method using the concept of Discriminating Power by Moffat et al. [45,46]. They found that the probability of discriminating between two compounds chosen at random in the data base was 99.67%, using a tolerance of 75 Kova´ts RI units. Rasanen et al. [47] evaluated their dual-column method consisting of separation on DB-5 and DB-1701. Although a high correlation (0.990) was found between the retention data, suggesting an apparent similarity between these columns, the correlation coefficient is not a sufficiently sensitive measure to judge the feasibility of using two columns in parallel. Instead, statistical calculations, based on the Mean List Length method [48], show that a 30–57% improvement in the identification power can be obtained using the dual-column approach at analytically relevant standard deviation (SD) levels. Interestingly, the highest gain is in the SD range of 4–8, and this decreases as the SD increases or decreases. An average intralaboratory SD of 1 was obtained for the present RI method during the 12-week period. In interlaboratory use, an SD value of 5–10 can be expected for a RI method [49]. References pp. 423–424

Chapter 11

410 11.4 IDENTIFICATION 11.4.1 Retention parameters

High precision of the appropriate retention parameter is crucial in GC screening, in contrast to GC-MS, where broader tolerance may be allowed. Recently, the status of retention-based identification has been restored by advances in GC technology, leading to approaches like fast chromatography and method translation [7,50]. Three types of retention parameters have been used for identification in GC. The absolute retention time (RT), or the adjusted retention time (RT’), is the most imprecise identification method. Variations in carrier gas flow rate, oven temperature and film thickness influence the RT. However, the RT is simple to measure and may be useful in solving trivial identification problems. Retention related to a single standard, the relative retention time (RRT), compensates for the effects mentioned above and allows more precise identification. However, the RRT is a retention parameter that is at its best in intralaboratory use under exactly the same instrumental conditions [51]. Obviously, the most precise identification parameter in GC is the retention index (RI). The RI system was originally based on the theory of Kova´ts [52]. The retention indices are calculated using at least two reference compounds that bracket the analytes. In GC drug screening methods, the aim is usually either to obtain Kova´ts indices using n-alkanes as RI standards, which can be compared with reference values in large databases using a search window of 725–60 units [44], or to obtain more precise retention parameters for daily intralaboratory use. The latter approach necessarily involves the use of non-alkane standards that are detectable by selective detectors and are structurally similar to analytes [49,53]. The RI methods reported in the literature predominantly use the external standard method; the standard series is injected daily before or between a sequence of analysis samples. The internal standard method, on the other hand, involves the injection of the standard series with each analysis sample.

11.4.2 Retention index systems According to Kova´ts [52], the plot of the logarithm of retention volume versus the carbon number of a homologous series is linear under isothermal gas chromatographic conditions. Later, it was suggested that retention volumes can be replaced by adjusted retention times [54]. Kova´ts’ equation for the calculation of retention index (RIx) is RIx ¼ 100n þ 100

log RT0 ðxÞ  log RT0 ðnÞ log RT0 ðn þ 1Þ  log RT0 ðnÞ

(1)

where RT0 (x) is the adjusted retention time of the unknown substance, RT0 (n) and RT0 (n+1) are the adjusted retention times of the n-alkanes, with carbon number n used as a standard as follows: RT0 (n)oRT0 (x)oRT0 (n+1).

Forensic screening by gas chromatography

411

In linear temperature programming, the logarithms of adjusted retention times can be replaced by absolute retention times (RT) [55] and equation (1) can be rewritten as RIlx ¼ 100n þ 100

RTðxÞ  RTðnÞ RTðn þ 1Þ  RTðnÞ

(2)

where RIlx is the linear retention index of x. However, for any homologous series of aliphatic organic compounds, there is a strong deviation from linearity in both cases for the first members of a series [54]. The temperature programs of instruments may deviate considerably from linearity, especially at the beginning and the end of the program, and the dependence of carrier gas flow upon temperature further enhances the non-linearity of the retention times of a homologous series. To compensate for these errors, cubic spline interpolation can be used. Cubic splines are functions composed of third-order polynomials. Linear retention indices may be calculated when using a non-linear temperature program, but more correct Kova´ts retention indices compared with linear behaviour can be calculated by using cubic splines [56]. Kova´ts indices can also be predicted from chemical structures by using chemometric methods. Garkani-Nejad [57] reported a standard error of prediction of about 80 RI units for a set of 846 organic compounds relevant in forensic analysis. This approach may be useful in confirming GC-MS results that are based on MS library search data only, when the reference standard is not available. 11.4.3 Retention index standards Identification in drug screening is often based on RIs, with n-alkanes as standards, and comprehensive drug retention index libraries are available. Although the libraries can be used on an interlaboratory basis for tentative identification of unknown drugs, the daily precision obtained by this method is low. In drug screening, medium polar stationary phases are preferred, and the retention of non-polar n-alkanes is reduced, which may result in the comparison of lowboiling polar solutes with n-alkanes of much higher boiling points. The high-boiling n-alkanes may require a significant increase in injector temperature, which can result in the formation of artefacts from temperature-sensitive sample components. Another problem is the weak response to n-alkanes of the selective detectors, mainly the nitrogen/phosphorous detector (NPD) and electron capture detector (ECD), required in drug analysis [51,54]. In temperature-programmed runs, the distribution constants and thereby the retention indices of different types of compounds are differently affected by changes in temperature. Small irregularities in a temperature program may thus lead to erroneous data. By using reference compounds with structures more similar to those of the samples, problems associated with differences in distribution constants can be minimized. Index standards should be stable both in solution and under the chromatographic conditions used. They should also bracket the solutes so that extrapolation is not required [33,54]. References pp. 423–424

Chapter 11

412

For drug screening, RI standards more similar to drugs in chemical nature have been suggested, including diisopropylamines [49], trialkylamines [58], nitroalkanes [59], alkylbis(trifluoromethyl)phosphine sulphides [36] and drug substances [33,60]. Franke et al. [49] found that drug substances, if selected carefully, were superior to the three first mentioned homologous series in terms of interlaboratory reproducibility. Rasanen et al. [61] proved that the internal standard RI method was regularly more precise than the external standard RI method. To avoid co-injection of commercially available drugs, they synthesized several dedicated homologue RI standard series for benzodiazepines [62], basic drugs [63] and acidic/neutral drugs [64] and demonstrated that the precision based on these internal standards was better than that of the external drug series. 11.4.4 Retention time locking (RTL) Recently, the concepts of method translation and RTL in GC were introduced by Blumberg and Klee [7]. These are based on void time being a universal time unit in GC, and method translation being the scaling of the time axis of the temperature program relative to the void time. Method translation can be used for RTL, which allows chromatograms to be reproduced accurately from one GC to another or over a long period of time [65]. RTL has been successfully applied to multiresidue screening of pesticides in fruit and vegetable extracts by matching GC and GC-MS retention times to a common database [66]. Fig. 11.1 compares the long-term precision obtained by using three different retention parameters on HP-5 and DB-17 columns [41]. The retention parameters studied were RT, RRT related to dibenzepin and the internal RI based on the alkylfluoroaniline series [63]. The carrier gas program was set in the constant flow mode. The drug substances represented various secondary and tertiary aliphatic amine HP-5

DB-17

0.90

0.90 0.80

0.70

0.70

0.60

0.60

Mean CV%

Mean CV%

0.80

0.80

0.50 0.40 0.30

0.84

0.50 0.40 0.30 0.30

0.21

0.21

0.20 0.10

0.20 0.09

0.10 0.06

0.05

0.00

0.10

0.14

0.13 0.06

0.00

RT RRT 1 RI Without RTL (left) and with RTL (right)

RT RRT 1 RI Without RTL (left) and with RTL (right)

Fig. 11.1. Precision (CV%) of the retention parameters on HP-5 and DB-17 columns without and with retention time locking (RTL).

Forensic screening by gas chromatography

413

structures. All results were based on 128 repetitive runs of spiked bovine blood extracts during an 18-week period. A clear improvement was observed in the precision (CV%) of all three retention parameters on both columns using the RTL function, the benefit being largest with RT and smallest with RI. While all RTL-based retention parameters showed very high precision without large differences, RRT with an average CV below 0.1% on each column was in general the most precise approach for drug screening. The positive results obtained by RTL suggest that no column selectivity change caused chromatographic variation due to loading with biological extracts, as this could not be efficiently compensated by RTL. 11.5 DETECTION AND QUANTIFICATION 11.5.1 Sample introduction Sample introduction into GC has a decisive influence on RT, LOD and quantitative results of analysis. For high precision, use of an autosampler is recommended. To obtain sufficiently low LODs with tolerable contamination of the analytical column, the splitless injection is generally used. Both the injection liner geometry and the position of glass wool to facilitate evaporation inside the liner may have a significant effect on the results. In addition, proper deactivation and cleaning of the liner and replacement of the glass wool at regular intervals are necessary. Choice of injection solvent, injection volume and initial column oven temperature should be carefully considered. The theory and practice of split and splitless injection have been thoroughly investigated by Grob [67]. 11.5.2 Detection Flame-ionization detector (FID) has a nearly universal response to organic compounds, a low LOD and a wide linear response range (107). The FID response results from the combustion of organic compounds in a small hydrogen-air diffusion flame. It is the most popular GC detector in current use. In drug screening, however, there may be interference from biological background, and this may be pronounced with post-mortem samples. In the absence of reference standards, FID can be used to predict relative response factors of known structures with reasonable accuracy using the effective carbon number concept, as shown with amphetamine-type compounds by Huizer et al. [68]. NPD is identical to FID, except that an alkali metal salt source is placed between the burner tip and the collector. Using low hydrogen flow rates, NPD is selective to nitrogen, having 104–105 times the response relative to carbon, and has a moderate linear response range (105). Tables 11.1 and 11.2 show that NPD is the most popular detector in toxicological drug screening. Most nitrogen-containing drugs give a satisfactory response, but nitro-compounds, amides and carbamates, such as meprobamate, are less favourable. Both the selectivity and the sensitivity of the detector are dependent on experimental variables, including the source heating current, References pp. 423–424

414

Chapter 11

source location, jet potential, air and hydrogen flow rates and choice of carrier gas. Especially the alkali bead lifetime may vary markedly depending on the individual bead and on the samples. With a workload of 30 runs per day in the authors’ laboratory, the beads from a major manufacturer lasted from two weeks to four months. NPD is not compatible with silylation reagents and halogenated injection solvents such as dichloromethane. Surface ionization detector (SID) is based on a similar principle to NPD, but it is substance-selective rather than element-selective. According to Kageura et al. [69], SID exhibited a high response for tertiary amines, a rather low response for secondary amines, no response for amides and little or no response for xanthines and benzodiazepines. Despite its potential in forensic toxicology, SID has not been used in comprehensive GC screening for drugs. Electron capture detection (ECD) can be used for the sensitive analysis of compounds that have high electron affinities. Electrons produced from a radioactive 63Ni source are selectively captured by, e.g., pesticides and drugs with certain structures, such as a halo group or the nitro group, or with lesser sensitivity, the carbonyl group. ECD is commonly applied to toxicological screening for benzodiazepines [34,40], but it produces no response with the 7-amino derivative of nitrazepam. The detector’s linear response range is somewhat limited (104), which may result in a need to dilute the sample. MS detection has, to a certain extent, replaced the detectors described above due to its unsurpassed properties in structural analysis. However, routine target screening of 50–200 compounds is often more feasible using selective detectors such as NPD and ECD. These detectors become even more attractive when comprehensive screening and simultaneous quantification are required at low concentrations in blood. In MS, the number of compounds that can be analysed in one run using selected ion monitoring (SIM) has been restricted, and the full scan mode may in turn lack sufficient sensitivity. This dilemma may be solved by splitting the GC-MS column effluent to a second selective detector like NPD.

11.5.3 Quantification GC screening is amenable to simultaneous quantification. To maintain a comprehensive quantitative screening of over 100 compounds, one-point calibration once a month has been deemed practical. Compounds that do not appear frequently can be quantified as required. The calibration concentration in one-point calibration can be set at the borderline between therapeutic and toxic levels to produce the best performance at the range of interest. Target screenings aiming for low concentrations usually require multi-point calibration. As shown in Tables 11.1 and 11.2, LOD of basic drugs is generally around 0.1–0.2 mg/l without derivatization. More precisely, as presented in Table 11.3, LOQ for aliphatic tertiary amines is 0.05–0.1 mg/l, and for aliphatic secondary and primary amines it is 0.2–0.5 mg/l. To detect and quantify the latter substances at therapeutic levels, derivatization is required. Lillsunde et al. [21] obtained an LOD of 0.01–0.1 mg/l for basic drugs in blood by GC-NPD/ECD after acylation with

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415

TABLE 11.3 RETENTION AND QUANTIFICATION DATA FOR 124 BASIC DRUGS [41] Compound Amitriptyline Amphetamineb Biperiden Bisoprolol Brompheniramine Bupivacaine Buspirone Caffeine Carbamazepinec Chlordiazepoxidec Chloroquine Chlorpheniramine Chlorpromazine Chlorprothixene Cinchocaine Cinnarizine Citalopram Clobutinol Clomipramine Clozapine Cocaine Codeine Cyclizine Dextrometorphan Dextropropoxyphene Diacetylmorphine Diazepam Dibenzepind Diltiazem Diphenhydramine Disopyramide Doxapram Doxepin Ethylmorphine Fencamfamin Fenfluramine Fentanyl Flecainidec Fluconazole Flumazenil Fluoxetine Fluvoxamine Haloperidol Hydrocone Hydroxychloroquine Hydroxyzine Imipramine Ketamine Ketobemidone

References pp. 423–424

RRT HP-5

RRT DB-17

LOQ (mg/l)a

0.800 0.216 0.859 0.904 0.734 0.865 1.464 0.562 0.894 1.045 1.094 0.667 1.028 1.030 1.145 1.330 0.961 0.530 0.966 1.221 0.806 0.943 0.680 0.757 0.792 1.114 0.990 1.000 1.261 0.584 1.035 1.230 0.825 0.971 0.484 0.259 1.155 0.829 0.736 1.057 0.577 0.589 1.265 0.994 1.237 1.221 0.821 0.577 0.704

0.797 0.211 0.836 0.860 0.737 0.841 1.615 0.629 0.985 1.054 0.990 0.660 0.981 0.943 1.040 1.312 0.922 0.490 0.926 1.235 0.848 0.966 0.679 0.757 0.763 1.026 0.997 1.000 1.314 0.573 1.000 1.219 0.837 0.973 0.450 0.218 1.061 0.782 0.791 1.059 0.526 0.529 1.235 1.011 1.176 1.177 0.823 0.604 0.729

0.1 0.5 0.1 1.0 0.05 0.2 0.05 0.5 1.0 0.2 0.2 0.05 0.05 0.1 0.1 0.05 0.1 0.2 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.1 0.05 0.1 0.2 0.05 0.05 0.2 0.1 0.05 0.2 1.0 0.1 0.1 1.0 0.2 0.05 0.1 0.2

Chapter 11

416 TABLE 11.3 CONTINUED Compound Levomepromazine Lidocaine Maprotiline MDMA Meclozine Melperone Mepivacaine Mesoridazine Metamphetamine Methadone Methyl phenidate Metoclopramide Metoprololb,c Mexiletine Mianserine Milnasipram Mirtazapin Moclobemide Molindone Moperone Nefazone Nicotine Nomifensine Norcitalopram Norclomipramine Nordazepam Nordextropropoxyphene amide Nordoxepin Norlevomepromazine Normethadone Normianserine Norpromazine Nortramadol Nortrimipramine Nortriptyline Norverapamilb Noscapine Olanzapine Orphenadrine Oxycone Pentazocine Pentoxyverine Pethidine Phenazone Phencyclidine Pheniramine Phentermine Phenytoin Pholcodine

RRT HP-5

RRT DB-17

1.050 0.590 0.908 0.406 1.295 0.597 0.712 1.547 0.237 0.762 0.513 1.107 0.684 0.337 0.807 0.931 0.840 0.834 1.020 1.211 2.045 0.313 0.769 0.982 0.987 1.037 1.065 0.837 1.069 0.732 0.841 0.922 0.647 0.840 0.813 1.421 1.381 1.159 0.626 1.047 0.859 0.838 0.520 0.582 0.599 0.554 0.229 0.916 1.380

0.995 0.577 0.907 0.398 1.252 0.557 0.728 1.779 0.225 0.743 0.513 1.087 0.666 0.321 0.844 0.862 0.885 0.877 1.023 1.138 e

0.309 0.850 0.952 0.958 1.044 1.020 0.870 1.025 0.723 0.900 0.944 0.662 0.850 0.834 1.541 1.569 1.140 0.612 1.046 0.849 0.800 0.500 0.664 0.565 0.551 0.219 0.982 1.532

LOQ (mg/l)a 0.1 0.1 0.2 0.2 0.05 0.05 0.2 0.2 0.2 0.05 0.05 0.05 0.5 0.5 0.05 0.1 0.05 0.1 0.1 0.1 0.5 0.1 0.2 0.5 0.5 0.1 0.2 0.2 Qualitative 0.1 0.1 Qualitative 0.1 0.2 0.1 1.0 0.2 0.05 0.1 0.1 0.1 0.1 0.1 0.5 0.05 0.05 0.05 5.0 0.5

Forensic screening by gas chromatography

417

TABLE 11.3 CONTINUED Compound Prilocaine Procainamide Promazine Promethazine Propranololb,c Quetiapine Quinine Reboxetine Ropivacaine Selegiline Sertraline Strychnine Temazepamc Thioridazine Thioridazine, 5-sulfoxide Tizanidine Tramadol Tramadol, O-desmethyl Trazodone Trimeprazine Trimetoprim Trimipramine Venlafaxine Verapamil Zaleplon Zolpidem Zopicloneb,c

RRT HP-5

RRT DB-17

LOQ (mg/l)a

0.567 0.830 0.900 0.862 0.768 1.425 1.201 0.913 0.802 0.353 0.929 1.376 1.229 1.357 1.723 1.011 0.630 0.675 1.501 0.880 1.082 0.817 0.719 1.381 1.297 1.202 1.343

0.559 0.893 0.909 0.879 0.775 1.604 1.187 0.942 0.793 0.335 0.918 1.593 1.340 1.438

0.1 5.0 0.1 0.1 0.5 0.2 0.2 0.5 0.1 0.1 0.1 0.1 0.2 0.1 0.2 0.5 0.1 0.2 0.2 0.1 1.0 0.1 0.1 0.1 0.1 0.1 0.02

e

1.081 0.620 0.688 1.732 0.878 1.096 0.803 0.712 1.445 1.416 1.228 1.555

a

Criteria for LOQ: 20% precision and 15% accuracy in the quantitative result of four parallel samples using single-point calibration.

b

Quantitation preferably by another dedicated method. Several peaks produced; the main peak is indicated here. d Internal standard. e Not analysed on this column. c

heptafluorobutyric anhydride. Benzodiazepines can be quantified at 0.006–0.075 mg/ l by GC-ECD after silylation [40].

11.6 ESTABLISHED GC SCREENING FOR BASIC DRUGS 11.6.1 General In the following, an established GC method is described that has been in routine use in the authors’ laboratory for several years [41]. The method serves as a backbone of basic drug screening and quantification for both post-mortem and clinical samples. Target analyses by other techniques, such as GC-MS and LC-MS, are used to References pp. 423–424

418

Chapter 11

supplement the GC screening. Although developed for blood (whole blood, plasma, serum), the present GC screening is also useful in the analysis of urine. LC-based techniques are, however, preferable in urine analysis, as they are better suited to deal with the polar substances excreted into urine. 11.6.2 Sample preparation Whole blood (1 ml) was transferred to a narrow centrifuge tube, Tris-buffer (1 M, pH 11, 0.3 ml) and the internal standard (dibenzepin 20 mg/ml in MeOH, 50 ml) were added and the mixture was shaken. The sample was extracted with butyl acetate (0.3 ml) in a multi-tube vortex mixer, centrifuged and an aliquot of the organic phase (150 ml) was transferred to an autosampler vial. 11.6.3 Gas chromatography The GC was equipped with two parallel fused silica capillary columns, HP-5 and DB-17 (15 m
0.32 mm, inner diameter, 0.25 mm film thickness), and two NP detectors (3301C). Uncoated deactivated fused silica precolumns of 10 m
0.32 mm were connected to the analytical columns. The precolumns entered a single injector (2701C) through a dual column injector adapter. A deactivated straight liner with silanized glass wool was used in the injector. Automated injections were performed using a 2-ml apparent injection volume. The carrier gas was helium, operated in the constant flow mode. The oven temperature was initially held at 1001C for 0.4 min, then increased by 251C/min to 2001C, increased by 101C/min to 2401C and increased by 251C/min to 2901C, where it was held for 10 min. The carrier gas flow was 2 ml/min for 15 min and then increased by 2 ml/min2 to 4 ml/min, which was held for 6.4 min. Under these conditions with new analytical columns and new 10-m precolumns, the RT of dibenzepin was 9.2 min on HP-5. This setting was locked by using five-point calibration data obtained with nominal initial pressure and with pressures of 20%, 10%, +10% and +20% from the nominal initial pressure. Relocking based on one scouting run was performed daily. 11.6.4 Data processing The GC was operated and data were collected, integrated and saved using ChemStation software equipped with Retention Time Locking software (Agilent Technologies, Palo Alto, CA, USA). Data processing was performed by SC Chrombooster software (Sunicom, Helsinki, Finland). 11.6.5 Performance Table 11.3 shows the RRT and LOQ values obtained by single-point calibration for 124 basic drugs and metabolites on HP-5 and DB-17. This list includes most

Forensic screening by gas chromatography

References pp. 423–424 Fig. 11.2. A dual-column chromatogram obtained from an autopsy blood sample. Columns: HP-5 (upper) and DB-17 (lower).

419

Chapter 11

420 Data File Method Date Created Date Analyzed Description Channel Library

: 2022025.dta : basic.MTD : Thursday, May 4, 2006 at 23:58:43 : Friday, May 5, 2006 at 16:22:52 : Sample Name :

:1 :Basic1.LBY

Peak Compound 1 methamphetamine 4 mexiletine 5 selegiline 6 fencamfamin 7 caffeine 8 tramadol 9 nortramadol 11 cyclizine 12 normethadone 13 nomifensine 14 normianserine 15 pentazocine 16 normirtazapin 17 carbamazepine 18 codeine 19 ethylmorphine 20 dibenzepin/INT.STD 21 trimethoprim

AbsRT IdPara/Mtd 2.573 3.366 3.559 4.660 5.393 6.029 6.207 6.460 6.928 7.311 7.941 8.059 8.224 8.382 8.805 9.030 9.209 9.807

0.279 R 0.366 R 0.386 R 0.506 R 0.586 R 0.655 R 0.674 R 0.701 R 0.752 R 0.794 R 0.862 R 0.875 R 0.893 R 0.910 R 0.956 R 0.981 R 9.209 A 1.065 R

Diff 0.00164 0.00420 -0.00283 -0.00216 -0.00114 -0.00111 -0.00376 0.00257 0.00150 -0.00299 -0.00063 0.00322 -0.00204 0.00156 -0.00009 -0.00156 0.000 0.00068

Total Channel Library

mg/l

2.92 20.95 1.68 48.28 34.43 112.86 8.88 8.14 4.24 10.94 77.81 1.74 5.98 6.90 21.31 1.78 110.45 28.87

0.068 0.423 0.009 0.785 1.334 1.528 0.375 0.055 0.062 0.160 1.536 0.043 0.000 0.438 0.973 0.059 1.000 1.834

508.16

10.680

:2 :Basic2.LBY

Peak Compound

5 MDMA 7 ketamine 8 tramadol 9 caffeine 10 phenazone 11 o-desmethyltramadol 14 dextromethorphan 15 amitriptyline 16 moclobemide 17 mirtazapin 18 normianserine 19 reboxetine 20 dibenzepin/INT.STD 21 trimethoprim 22 pholcodine Total

Area

AbsRT IdPara/Mtd 3.851 4.805 7.143 7.365 7.478 7.838 8.137 8.853 9.203 9.863 9.977 10.118 10.436 11.116 12.388 16.823

0.346 R 0.432 R 0.643 R 0.663 R 0.673 R 0.705 R 0.732 R 0.796 R 0.828 R 0.887 R 0.898 R 0.910 R 0.939 R 11.116 A 1.114 R 1.513 R

Diff 0.00262 -0.00132 0.00343 -0.00199 -0.00191 0.00012 -0.00275 -0.00017 -0.00125 0.00116 -0.00061 -0.00032 0.00057 1.000 0.00029 -0.00191

Area

mg/l

30.12 2.49 13.86 132.98 45.13 13.00 10.78 2.69 8.49 2.36 91.67 14.23 4.07 128.84 29.02 4.54

0.232 0.069 0.234 1.527 1.500 0.945 0.221 0.046 0.111 0.030 0.587 0.253 0.117 1.000 1.789 0.520

534.28

9.181

Fig. 11.3. Single-column reports from HP-5 and DB-17 for the case in Fig. 11.2, providing the best hit for each peak.

Forensic screening by gas chromatography

421

*** SC-Compare Report [Version 1.50] *** Data File Method Date Created Date Analyzed

: 2022025.dta : basic.MTD : Thu May 4 2006 at 23:58:43 : Fri May 5 2006 at 16:03:43

Compound Ch. Peak AbsRT IdPara Diff Area Amount ----------------------------------------------------------------------------------------------------------------tramadol 1 8 6.029 0.655 0.001 112.86 1.528 2 8 7.365 0.663 0.002 132.98 1.527 -----------------------------------------------------------------------------------------------------------------caffeine 1 7 5.393 0.586 0.001 34.43 1.334 2 9 7.478 0.673 0.001 45.13 1.500 ----------------------------------------------------------------------------------------------------------------nortramadol 1 9 6.207 0.674 0.003 8.88 0.375 0.462 2 10 7.838 0.705 0.003 13.00 ----------------------------------------------------------------------------------------------------------------0.190 6.460 o-desmethyltramadol 1 11 0.701 0.002 8.14 2 11 8.137 0.732 0.002 10.78 0.221 ----------------------------------------------------------------------------------------------------------------0.862 0.579 mirtazapin 1 14# 7.941 0.001 77.81 17 9.977 2 0.898 0.000 91.67 0.587 ----------------------------------------------------------------------------------------------------------------1.536 normianserine 1 14# 7.941 0.862 0.000 77.81 0.253 2 18 10.118 0.910 0.000 14.23 ----------------------------------------------------------------------------------------------------------------0.000 normirtazapin 1 16 8.224 0.893 0.002 5.98 0.000 2 19 10.436 0.939 0.001 4.07 ----------------------------------------------------------------------------------------------------------------1.000 dibenzepin/INT.STD 1 20 9.209 0.000 110.45 9.209 2 20 11.116 11.116 0.000 128.84 1.000 ----------------------------------------------------------------------------------------------------------------1.834 trimethoprim 1 21 9.807 1.065 0.000 28.87 1.789 2 21 12.388 1.114 0.000 29.02 ----------------------------------------------------------------------------------------------------------------Fig. 11.4. Advanced dual-column comparison report for the case in Fig. 11.3.

psychotropic and other prescription drugs relevant in forensic toxicology that can be analysed without prior derivatization by GC even at the therapeutic concentration level. Maintenance comprises changing the injector liner glass wool daily and the liner weekly, shortening the precolumns weekly by 50 cm and quantitative calibration at one-month intervals. References pp. 423–424

422

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Fig. 11.2 displays a typical pair of chromatograms obtained from an autopsy case, representing elevated blood concentrations of the opioid tramadol and the antidepressant mirtazapine, together with their metabolites. In addition, the antibacterial agent trimethoprim and caffeine are present at therapeutic levels. The single-column reports, providing the best hit for each peak, and the advanced dual-column comparison report for the above case are shown in Figs 11.3 and 11.4, respectively. These figures demonstrate that the dual-column comparison report makes the results legible by indicating only those substances for which preselected detection windows fit the detected peaks on both columns [47]. The only false- positive finding in this report, normianserin, can be ruled out by the large concentration difference between the columns and by recognizing that the peak on column one is reserved for mirtazapine (marked with #). Qualitative performance of the method is shown in Fig. 11.1, indicating excellent day-to-day precision of RRT (CVo0.1%). Quantitative performance of the method is sufficient for most applications of forensic toxicology. The LOQ, typically ranging from 0.05 to 0.2 mg/l, is adequate for analysing most basic drugs at therapeutic levels (Table 11.3). The expanded uncertainty of measurement generally lies between 15% and 40%. Interlaboratory proficiency testing has been carried out with amitriptyline, citalopram, dextropropoxyphene, levomepromazine (methotrimeprazine) and methadone, all of which show good z-score values, always below 1.4 units (deviation from distribution’s mean, expressed in units of SD) over a four-year period. The method has been accredited by the Finnish Centre for Metrology and Accreditation (FINAS).

11.7 CONCLUSIONS Broad-scale screening for drugs and poisons is an integral part of forensic and hospital toxicology. The aim of drug screening is to identify and preferably also to quantify a maximal number of potentially toxic compounds present in a biological sample. Despite the increased attention directed at GC-MS and LC- MS techniques, GC screening with selective detection has maintained its position as a rapid and cost-effective tool for analysing the majority of common drugs involved in poisonings. Especially the dual-column approach, consisting of two parallel columns of different selectivity and two similar detectors, provides a high identification power close to GC-MS. Comparison of the quantitative response factors on the two columns provides an additional identification parameter. Applying RTL function significantly improves the precision of identification, not only between systems or laboratories, but also within an individual GC instrument and column. In addition, GC usually allows more substances to be included in quantitative analysis than GCMS in the SIM mode. As 90% of drugs contain nitrogen, NPD is indispensable for detecting these substances, with minimal interference from the matrix. Particularly for tertiary amine drugs, GC-NPD can still be considered the optimal choice. Finally, the success of GC screening is very much dependent on the performance of the reporting software, especially in dual-column operation.

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423

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