Review Special Focus Issue: Forensic and clinical toxicology

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The current role of on-line extraction approaches in clinical and forensic toxicology

In today’s clinical and forensic toxicological laboratories, automation is of interest because of its ability to optimize processes, to reduce manual workload and handling errors and to minimize exposition to potentially infectious samples. Extraction is usually the most time-consuming step; therefore, automation of this step is reasonable. Currently, from the field of clinical and forensic toxicology, methods using the following on-line extraction techniques have been published: on-line solid-phase extraction, turbulent flow chromatography, solid-phase microextraction, microextraction by packed sorbent, single-drop microextraction and on-line desorption of dried blood spots. Most of these published methods are either single-analyte or multicomponent procedures; methods intended for systematic toxicological analysis are relatively scarce. However, the use of on-line extraction will certainly increase in the near future.

In today’s clinical and forensic toxicological laboratories, automation is of interest for several reasons. By increasing the degree of automation for complex systems, such as hyphenated techniques (e.g., liquid chromatography–mass spectrometry [LC–MS] or gas chromatography–mass spectrometry [GC–MS]), processes can be optimized, manual workloads may be reduced drastically and handling errors due to manual processes are minimized [1] . Moreover, the handling of potentially infectious samples is much safer when the manual steps are omitted [2] . In comparison with related laboratory disciplines (e.g., clinical chemistry), the degree of automation in clinical and forensic toxicological laboratories is usually much lower. The first mechanized clinical–chemical method was published in 1957 [3] , with the first automated tests (creatinine and uric acid) following shortly afterwards [4] . By contrast, the use of automation techniques in clinical and forensic toxicology (e.g., on-line extraction) only begun in recent years. Clinical and forensic toxicological laboratories deal with complex biological matrices such as serum, whole blood, urine or hair. Therefore, sample preparation is manda-

10.4155/BIO.14.179 © 2014 Future Science Ltd

Daniel M Mueller Institute for Clinical Chemistry, University Hospital Zurich, Raemistrasse 100, 8091 Zurich, Switzerland Tel.: +41 44 255 22 90 Fax: +41 44 255 45 90 [email protected]

tory before analysis with modern analytical techniques. These preparation steps, usually also involving extraction steps, are often very time consuming: approximately 80% of the analysis time is spent with sample preparation and sample extraction [5,6] . With online extraction, the total analysis time can be reduced drastically, because no manual extraction step is necessary [6] . Moreover, sample preparation and extraction steps require many manual handling steps that are prone to errors and also induce variability. Therefore, the automation of extraction steps makes a lot of sense for clinical and forensic toxicology. The higher initial costs and the higher complexity of the technical equipment needed for on-line extraction is discouraging. However, despite the more expensive acquisition costs, the running costs may be reduced due to the decreased use of consumables. Moreover, because manual handling time can be reduced drastically, on-line extraction systems can be cost-effective [7] . Several on-line extraction techniques are available. This article will focus on techniques for which papers with a clinical or forensic toxicological focus have been

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Key terms On-line extraction: Technique by which the extraction procedure is directly coupled to the analytical technique (e.g., liquid chromatography–mass spectrometry) without the requirement of manual sample-handling steps after the extraction process. Dried blood spot: Sampling technique in which blood is spotted onto a filter paper and dried.

published in English since the late 1990s. These are the following techniques: on-line coupled solidphase extraction (SPE), including on-line SPE using newer types of sorbents (e.g., restricted access materials [RAMs], monolithical sorbents, molecularly imprinted polymers [MIPs] and immunoaffinity sorbents); turbulent flow chromatography (TFC); solidphase microextraction (SPME); microextraction by packed sorbent (MEPS); single-drop microextraction (SDME); and on-line desorption of dried blood spots (DBSs). On-line extraction techniques used in clinical & forensic toxicology On-line SPE

The most abundant technique is on-line coupled SPE. On-line SPE uses a dedicated extraction column, cartridge or SPE disc to trap the analyte(s). There are systems with two dedicated pumps available (loading and eluting). More simple systems include only one pump, which is used for both the extraction as well as the analytical chromatography [8] . With two-pump systems, the extraction column can be preconditioned during the run time of the analytical chromatography, which can be used to optimize the throughput. Commercial apparatus of both configurations are available, making the technique easily available to a broad range of clinical and forensic toxicological laboratories. A good overview of recent developments in this area is given by Rogeberg et al. [8] . In clinical and forensic toxicology, wide concentration ranges of analyte(s), sometimes in the range of several orders of magnitude, may occur in the analyzed samples (e.g., after a fatal intoxication case). With regards to this fact, on-line SPE has an inherent advantage of preventing carry-over: it has the potential for changing the extraction cartridge or disc for each sample. This advantage is shared with other techniques (e.g., MEPS systems using sorbent packed in pipetting tips, as well as SDME). With on-line SPE, the whole sample is analyzed, not only an aliquot after extraction. Therefore, sensitivity is optimized, as there is no loss due to concentration or dissolution steps, and sample volume can be minimized [9,10] .

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Compared with off-line SPE, the restricted use of extraction solvent is a drawback of the on-line variant. Compromises are necessary concerning the compatibility of the extraction solvents with the subsequently coupled analytical technique, which is usually LC–MS. An example is the use of strong alkaline elution solvents for the elution of the analytes from strong cation exchange columns in off-line SPE. These strong alkaline solvents are not compatible with LC–MS analysis [11] . Another drawback of on-line SPE is the risk of clogging of the column; this is sometimes a limiting factor for the usage of on-line SPE systems in routine applications [8] . In clinical and forensic toxicology, the sorbents that are most often used for on-line SPE are reversedphase bonded silica or polymer resins. However, newer types of sorbents are also used (e.g., RAMs) [12] . Usually, materials with small pores are used to achieve a size exclusion effect. RAMs may also be used with different chromatographic interaction modes for particles that are small enough to be able to diffuse into the pores [13] . Another example of newer types of sorbents are monolithical supports. The main advantages of this material include its stability over a wide range of pH values and its low back pressure, depending on the very porous nature of the structure [14] . MIPs are polymers containing cavities that selectively recognize the analyte(s) of interest [15] . The main advantage of this technique is therefore its selectivity. However, in clinical and forensic toxicology, broader selectivity is sometimes a must (e.g., for screening approaches). MIPs are less suitable for these types of approaches. MIPs may also be coupled with RAMs [16] . MIPs are commonly not compatible with direct injection of biological matrices: they exhibit hydrophobic polymer surfaces that also retain proteins [17] . Therefore, additional techniques must be applied to remove proteins and other potentially interfering compounds before transferring them to the analytical column (e.g., by the combined use of RAMs and MIPs) [15] . Related to MIPs are immunoaffinity columns, which do not use cavities but rather use antibodies to selectively retain the wanted analyte or class of similarly structured analytes [18] . The same advantages and disadvantages as for MIPs apply regarding the selectivity of immunoaffinity columns. On-line SPE was compared with traditional offline liquid–liquid extraction (LLE) for the analysis of psilocin in human plasma. The analytical performance was comparable between both extraction techniques. However, when using on-line SPE, a substantially reduced sample volume was needed in order

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The current role of on-line extraction approaches in clinical & forensic toxicology

to reach the required lower limit of quantification, demonstrating the higher sensitivity achievable by using on-line extraction. Furthermore, less manual workload was necessary when using on-line SPE [19] . An interesting paper comparing on-line SPE using different sorbents (including a RAM sorbent), offline SPE, LLE and protein precipitation (PPT) using methadone as a model compound was published by Souverain et al. [20] . The authors concluded that LLE was the extraction procedure exhibiting the fewest matrix effects, while PPT resulted in the most signal suppression. For on-line SPE, no matrix effects were observed for one of the tested on-line SPE sorbents, and only a minor effect – comparable to the off-line SPE procedure – was observed for the RAM sorbent. A significant matrix effect was detected for the third sorbent that was studied, indicating that careful choice of the extraction sorbent as well as the method of optimization is most important when using on-line SPE. Turbulent flow chromatography

TFC is the second most frequently used on-line extraction technique in clinical and forensic toxicology. TFC uses a turbulent rather than a laminar flow profile. The turbulent flow is generated by using high flow rates (>1.2 ml/min) in combination with large porous particles (30–50 μm) [7] . Due to the turbulent flow conditions, efficient mass transfers between the mobile phase and the pores of the spherical particles are achievable. TFC uses a size exclusion mechanism: small organic molecules (e.g., drugs) can efficiently diffuse into the pores and are therefore retained on the column. Larger matrix constituents (e.g., plasma proteins), which cannot diffuse inside the pores, are eluted to the waste with the exclusion volume of the column [21] . The inside of the pores of the particles can also be coated with different chromatographic specificities (e.g., C18), which also contributes to the retention of the analytes that can diffuse into the pores. Using different chromatographic specificities, the selectivity of the extraction may be varied [21] . An important advantage of TFC is the high purity of extracts that is achievable with this method. This was shown in several publications: TFC extracts caused fewer interferences in the the analyses of αand β-amanitin compared with PPT, SPE and LLE [7] . TFC was directly compared with PPT using a method for the quantification of nicotine and two of its metabolites in serum [10] . After PPT, severe matrix effects were observed, which could be eliminated by using TFC as an additional extraction technique. Zimmer et al. described a very extensive method comparison between LLE, semiautomated SPE using

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96-well plates and TFC [6] . Using two structurally different compounds, they concluded that the results of the TFC method were similar to the reference LLE method and slightly better than the semiautomated SPE method. TFC uses the same extraction column for several samples fir up to several hundred or even several thousand samples; extraction costs per sample are very low. However, regarding carry-over, this technique does not show the same inherent safety as, for example, on-line SPE using new extraction columns for every sample. Carry-over must therefore be evaluated very carefully [22] . In order to ensure optimal column lifetime, clogging should be prevented cautiously (e.g., by using in-line filters). Solid-phase microextraction

In SPME, a solid support consisting of a fused silica or metal fiber that is coated on the outside with a suitable stationary phase is used to adsorb the analytes. When using polymeric monolithic material as an adsorption material, this technique is called polymer monolith microextraction [23] . A good overview of this approach is given by Farhadi et al. [24] . In the most common technique, the extraction fiber is placed directly into the sample [24] . In in-tube SPME, a capillary is coated on the inside with a suitable stationary phase. The sample is flown through this capillary and the analytes adsorb to the inner surface of the capillary [24] . In both techniques, analytes are desorbed on-line. The main advantages of this technique are miniaturization, which leads to minimal demands on sample volume, and the elimination of solvents for extraction, which is beneficial for the environment as well as financially favorable. The disadvantages of this technique include the risk for carryover when using the same extraction support for several samples, the often low recovery and, depending on the matrix, the susceptibility of the fiber to physical damage. Furthermore, LLE and SPE were shown to be more precise techniques, and the quality of the fiber is said to differ significantly from batch to batch [25] . Microextraction by packed sorbent

In MEPS, the sorbent is packed either into a syringe [26] or into pipette tips [27] . The sample is aspirated into the syringe or flown through the pipette tips. A broad range of different sorbents may be used. If the sorbent is placed in a syringe that is used for more than one sample, careful validation of carry-over is mandatory. An advantage of this method is that it can be implemented easily into pre-existing systems; there is no need for additional robots [28] . Moreover, the technique is very favorable for miniaturization.

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Review  Mueller Single-drop microextraction

In SDME, a single drop of solvent (e.g., suspended from the tip of a syringe) is immersed in the sample. Analytes are extracted into this drop, which can subsequently be injected into the chromatographic or electrophoretic system [29] . Three-phase principles with a first extraction into an organic layer and subsequent back-extraction into an aqueous phase are also used [29] . The main advantage of this technique is the use of a very clean extraction technique: in different comparisons between LLE and SPE, LLE resulted in at least comparable extract purities as SPE [30–32] . However, SDME omits the main disadvantage of off-line LLE, which is the high consumption of possibly toxic solvents. Until recently, a major disadvantage of this technique was its difficult handling; recently, however, relevant progress has been made [33–35] . It will take some time for these advances to be reflected in publications outside of the field of clinical and forensic toxicology. On-line extraction systems for DBS

Using two clamps, a mobile phase can be flown through a filter paper containing the DBS in order to extract the analytes. An interesting system that is able to handle whole filter paper cards is presented by Ganz et al. [36] . The use of DBS has been extensively reviewed recently [37] . In clinical and forensic toxicology, methods can be approximately separated into three basic groups: methods dealing with a single analyte and its metabolite(s); multianalyte procedures dealing with up to several tens of analytes; and screening analyses dealing with at least several hundred analytes, usable for systematic toxicological analysis. In the following section, an overview of on-line extraction methods outside of the field of clinical and forensic toxicology is given, which is divided into single analyte or multicomponent analyses and screening analyses. On-line extraction in single analyte & multianalyte analyses On-line SPE

An overview of the methods using on-line SPE that are covered in this article can be seen in Supplementary Table 1. In the following text, only publications of special interest are mentioned. Lindenblatt et al. described a method for the quantification of psilocin in plasma [19] . The extraction procedure using on-line SPE was compared with an off-line extraction procedure using LLE. The authors concluded that both extraction procedures resulted in similar analytical performances of the analytical methods. The advantages of on-line SPE were the higher degree of

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automation, better selectivity of the extraction and the lower sample volume required to reach the lower limit of quantification. The main disadvantage of on-line SPE was, according to the authors, the requirement for more complex apparatus. Krämer and Kovar described two different on-line SPE methods, one for the quantification of N-ethyl-4-hydroxy-3-­methoxyamphetamine (HMEA) in plasma and the other for the quantification of Δ9-tetrahydrocannabinol and 11-nor-Δ9carboxy-tetrahydrocannabinol in plasma and urine [38] . Several authentic clinical samples were analyzed in order to show the applicability of the method. As with most methods dealing with glucuronidated substances, manual hydrolysis of glucuronides was necessary prior to on-line SPE. The authors also chose to insert a manual PPT step in front of the on-line SPE, which was beneficial for column lifetime, but also increased the manual workload. Miki et al. published a method for the simultaneous quantification of methamphetamine and two of its metabolites in hair [39] . Due to the matrix, rather extensive manual sample pretreatment was necessary. The method was compared with a pre-existing GC–MS method. The authors concluded that their new on-line SPE–LC–MS method was more sensitive and faster than GC–MS, but otherwise comparable. In 2005, Wu and Fuh published a method for the simultaneous quantification of several amphetamines in urine [40] . Although urine was being used, the method required a manual PPT step and a very high sample volume. After the precipitation step, an aliquot of 10 μl was used for the analysis. Comparison to a previously used GC–MS method using five real-world samples revealed that both methods were comparable. In clinical toxicology, fast screening for the presence of tricyclic antidepressants is sometimes necessary. For this purpose, Cruz-Vera et al. published a method for fast urinary screening of two frequently used tricyclic antidepressants in urine [41] . The method used no analytical column; after derivatization, the analytes were detected by UV immediately following desorption of the on-line SPE cartridge. Robandt et al. published a paper describing the quantification of several metabolites of cocaine in human urine [42] . The method was completely automated using a liquid handler as well as on-line SPE. The method was compared with a pre-existing GC–MS method using LLE, and the authors’ conclusions were that both methods agreed well, but that the on-line SPE–LC–MS method was substantially faster: with the GC–MS method, 2 h were needed to process 25 samples, whereas the manual processing time was only 10–20 min for the same amount of sample using the newly developed method. Moreover, due to the omission of derivatization steps

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The current role of on-line extraction approaches in clinical & forensic toxicology

in LC–MS, the number of fume hoods could be reduced in the authors’ laboratory. Compared with similar on-line extraction methods, a disadvantage of the method was the rather high sample volume that was required for the described method. Ferreirós Bouzas et al. described a method for the simultaneous quantification of five opiates and their metabolites, five amphetamines and their metabolites, cocaine and its metabolite and methadone and its metabolite [43] . The authors state that by using a first PPT step, the lifetime of the columns could be extended. However, the PPT step had to be carried out manually, thus limiting the degree of automation of the method. As for many methods dealing with substances with a wide polarity range, matrix effects could not completely be excluded over the whole chromatographic run time; in this paper, ion suppression was observed for morphine and its glucuronides. Liao et al. described a method for the simultaneous quantification of two frequently used insecticides in Taiwan – chlorpyrifos and cypermethrin – in cord blood plasma [44] . As the extraction technique, on-line SPE was used. According to the authors, exposure to both pesticides imposed risks for the developing child. Because of the automation, the authors concluded that the method was suitable for high-throughput analysis of the 396 cord blood samples they analyzed. In 2011, Lacroix et al. described a method for the determination of cyanide in whole blood using on-line SPE [45] . In order to analyze a molecule as small as cyanide by reversed-phase LC, it had to be complexed with naphthalene-2,3-dicarboxyaldehyde and taurine. As a first step, whole blood had to be deproteinized and centrifuged manually, as is the case for most on-line extraction methods using whole blood. Afterwards, naphthalene-2,3-dicarboxyaldehyde and taurine were added, and the mixture had to be incubated for 10 min. Therefore, despite using online SPE, quite an extensive manual sample preparation was necessary. König et al. developed a method for the quantification of Δ9-tetrahydrocannabinol and two of its metabolites in peripheral blood [46] . The method was intended to be used in forensic toxicology in order to analyze suspected cases of driving under the influence of drugs. The authors stated that, due to the increasing number of cases, the capacity of their pre-existing method based on GC–MS was exceeded. Therefore, a more automated method using on-line SPE–LC–MS was developed. However, this method also requires a quite extensive manual sample pretreatment: samples were deproteinized and centrifuged and the supernatant was evaporated under a stream of nitrogen. After reconstitution, samples were ready to be analyzed by on-line SPE–LC–MS. De Jager and Bailey described a method for the simul-

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Key terms Systematic toxicological analysis: Systematic analysis of biological samples for the detection of the presence of drugs and chemicals of toxicological relevance.. Multicomponent analysis: The simultaneous analysis of several analytes, also from different classes, by a single analytical method. Glucuronide: Phase II metabolite of many substances, produced by coupling the parent substance or one of its phase I metabolites to glucuonic acid.

taneous quantification of 29 drugs of abuse [47] . The method required enzymatic hydrolysis of glucuronidated compounds, but otherwise, sample preparation consisted only of the addition of an internal standard and dilution solvent and, for enzymatically hydrolyzed samples, filtration. No centrifugation step was necessary according to the authors. Because urine, which is not analyzed within a few hours after sampling, often has particulate matter, it remains questionable as to whether the omitted centrifugation caused problems with the clotting of the columns, especially for the nonfiltrated native samples. Saussereau et al. developed a multianalyte procedure for the simultaneous quantification of 14 analytes (including opiates, cocainics and amphetamines) in DBSs, with subsequent on-line SPE [48] . The authors concluded that this method, because of its use of a matrix that can be easily stored and also mailed, is suitable for the analysis of suspected cases of driving under the influence of drugs, for example. DBS results were compared with results from fresh whole blood, demonstrating a good comparability. However, the inclusion of more analytes from different classes (e.g., cannabinoids) would have been beneficial. According to the authors, simultaneous quantification of cannabinoids would have needed dedicated extraction and chromatographic conditions. Bugey and Staub published a method for the quantification of eight benzodiazepines [2] . They compared a RAM for on-line SPE with a monolithical on-line SPE support. The method was fully validated using both on-line SPE materials. The authors described that the monolithical support performed better. Moreover, the robustness of the monolithical support was superior: after injection of more than 500 blood samples, no performance degradation was observed for the monolithical support, whereas significant increases of back pressure were observed for the RAM support after 400 injections. In 1999, Deinl et al. described an on-line SPE method for the quantification of flunitrazepam and its metabolites in plasma or serum [18] and urine [49] using immunoaffinity columns as extraction sorbents. The authors stated that the immunoaffinity columns could be reused several times with good stability: more

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300 μl serum

1 ml urine

Opiates (morphine, codeine and dihydrocodeine), benzoylecgonine, amphetamines (amphetamine, methamphetamine, MDMA, MDEA and MDA), EDDP and benzodiazepines (diazepam, nordazepam, oxazepam, hydroxyalprazolam, bromazepam, 7-aminoflunitrazepam and lorazepam)

Morphine, codeine, dihydrocodeine, oxycodone, oxymorphone, hydrocodone, hydromorphone, methadone, fentanyl, tramadol, propoxyphene, amphetamine, methamphetamine, cocaine and THCCOOH

Nicotine, cotinine and nornicotine

α-amanitin and β-amanitin

2012

2012

2013

2013

Cyclone + phenyl

MCX II

Cyclone MAX + Cyclone P

Cyclone MAX

C18XL

Yes (α-amanitin)

Yes

Yes

Yes

Yes

TFC column Validation

LC–MS

LC–MS

LC–MS

LC–MS

LC–MS

Analytical technique

Comparable with existing methods

4-min run time, comparable with existing methods

Besides hydrolysis of glucuronides, only minimal manual sample pretreatment

Besides hydrolysis of glucuronides, only minimal manual sample pretreatment

Minimal manual sample pretreatment

Advantages

[7]

[10]

Manual enzymatic hydrolysis of glucuronides [60]

Manual hydrolysis of glucuronides [50]

[59]

Disadvantages

EDDP: 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine; MDA: 3,4-methylenedioxyamphetamine; MDEA: 3,4-methylenedioxy-N-ethylamphetamine; MDMA: 3,4-methylenedioxymethamphetamine; TFC: Turbulent flow chromatography; THC-COOH: 11-nor-Δ9-carboxy-tetrahydrocannabinol.

200 μl urine

500 μl urine

100 μl plasma

Methoxetamine

2012

Sample volume and matrix

Analyte(s)

Year

Table 1. Single-analyte or multicomponent methods using turbulent flow chromatography.

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The current role of on-line extraction approaches in clinical & forensic toxicology

than 100 runs for plasma and more than 80 runs for urine were possible using the same column. Compared with other, more recent on-line extraction sorbents, this number is quite low for the studied matrices. The main advantage of the immunoaffinity column was its specificity for the analytes; the authors stated that no coextracted matrix constituents were detectable. Cross-reactivity with other benzodiazepines was tested. Chromatographic separation could be achieved in order to omit specificity problems. Turbulent flow chromatography

An overview of the methods using TFC covered in this article can be seen in Table 1. In the following text, only publications of special interest are mentioned. In 2012, Schaefer et al. published a paper describing two methods for the quantification of opiates, benzoylecgonine, amphetamines, 2-ethylidene-1,5-dimethyl3,3-diphenylpyrrolidine (EDDP) and benzodiazepines in urine [50] . As with most methods dealing with urine, manual enzymatic hydrolysis of glucuronides was necessary before the analysis. However, besides the addition of buffer and internal standards and centrifugation, no other sample preparation was necessary. The authors stated that the results obtained with the newly developed TFC–LC–MS method ‘differed slightly’ from the results of a references method (GC–MS). Yuan et al. published a method for the quantification of nicotine and two of its metabolites in serum samples [10] . With a total run time of approximately 4 min, the method was suitable for high-throughput analysis. Most likely, throughput is only limited by the manual PPT step required for the method. The

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authors described that after direct injection of the supernatant into the analytical column, severe matrix effects could be observed. These matrix effects could be eliminated by using TFC. Moreover, the method was compared with a previously used method using off-line SPE. The authors concluded that the sensitivity could be increased by using TFC with the same amount of sample volume. Helfer  et al. very recently published a method for the identification of α- and β-amanitin and the quantification of α-amanitin in urine [7] . The method was compared with a pre-existing ELISA method. The authors concluded that their method allowed for a fast and robust analysis of these two substances. Compared with LC methods using off-line extraction, TFC extracts caused less interference. Moreover, TFC was a cost-effective extraction technique for analyzing α- and β-amanitin. Compared with other TFC methods, this method uses a relatively high sample volume (1 ml), even more so when considering that only 100 μl is injected into the system. However, this demand on sample volume is similar to that of the pre-existing ELISA method. Solid-phase microextraction

An overview of the methods using SPME covered in this article can be seen in Table 2. In the following text, only publications of special interest are mentioned. Kataoka et al. described a method for the simultaneous quantification of nicotine, cotinine, nornicotine, anabasine and anatabine using in-tube SPME [51] . Sample preparation consisted of dilution of the samples. The authors concluded that they were able to achieve 20–46-times higher sensitivities compared

Table 2. Single-analyte or multicomponent methods using solid-phase microextraction. Year

Analyte(s)

Sample volume and matrix

SPME sorbent

Validation

Analytical Disadvantages technique

2000

Amitriptyline, nortriptyline, imipramine and desipramine

1.2 ml urine

Not described

Proof of principle

HPLC-UV

No validation [61]

2001

Amitriptyline, imipramine, nortriptyline, imipramine and desipramine

1 ml urine

Zylon® HM or AS

Proof of principle

CE-UV

No validation [62]

2006

Amitriptyline, nortriptyline, imipramine and desipramine

1 ml plasma

Polydimethylsiloxane- Proof of divinylbenzene principle

HPLC-UV

No validation [63]

2009

Nicotine, cotinine, nornicotine, anabasine and anatabine

100–200 μl urine or saliva

CP-PoraPLOT amine

Partial

LC–MS

Only partial validation [51]

2012

As(III), As(V), monomethylarsonic 1 ml urine acid, dimethylarsenic acid, arsenobetaine and arsenocholine

PSP/MPTS/AAPTS

Proof of principle

LC–ICP– MS

No validation [52]

AAPTS: N-(2-aminoethyl)- 3-aminopropyltrimethoxysilane; CE: capillary electrophoresis; LC–ICP–MS: LC-inductively coupled plasma-MS; MPTS: 3-mercapto propyltrimethoxysilane; PSP: Partially sulfonated poly(styrene).

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Table 3. Single-analyte or multicomponent methods using microextraction by packed sorbent. Year

Analyte(s)

Sample MEPS sorbent volume and matrix

Validation

Analytical technique

Advantages

Disadvantages

2004

Ropivacaine, PPX and 3-hydroxyropivacaine

1 ml

Silica-C2

Partial

LC–MS

–

Only partial validation and high sample demand [25]

2007

Methadone

1 ml

Silica-C8

Yes

GC–MS

–

No metabolites of methadone included and high sample demand [28]

2009

Cocaine, ecgonine methyl ester, benzoylecgonine and cocaethylene

Not specified

Silica-C8, ENV+, Partial Oasis MCX, Clean Screen® DAU®

MS

–

Only partial validation [26]

2010

Lidocaine, ropivacaine and bupivacaine

25 μl

Silica-C18

LC–MS

Very small sample demand

Information required to remove by peer review process (cf. remark in table 1) [53]

Yes

MEPS: Microextraction by packed sorbent; PPX: 2’,6’-pipecoloxylidide.

with direct injection. In 2012, Chen et al. published one of the very rare papers describing an on-line extraction method for the determination of arsenic species in urine [52] . The method was intended as a proof of principle for the use of in-tube SPME, coupled online to ion-pair liquid chromatography-iductively coupled plasma-mass spectrometry (LC–ICP–MS). The authors proved the applicability of this technique using the analysis of authentic urine samples. Microextraction by packed sorbent

An overview of the methods using MEPS covered in this article can be seen in Table 3. In the following text, only publications of special interest are mentioned. Jagerdeo and Abdel-Rehim described a method for the direct analysis of cocaine and three of its metabolites in urine samples [26] . After extraction using MEPS, the analysis was conducted directly with a time-offlight mass spectrometer without the usage of a chromatographic technique. Said et al. published a paper describing the quantification of three local anesthet-

ics in whole blood [53] . The method used only 25 μl, which is a very small sample volume, demonstrating the miniaturization potential of MEPS. Single-drop microextraction

An overview of the method using SDME covered in this article can be seen in Table 4. Gao et al. published a method for the simultaneous quantification of three alkaloids in human urine [29] . The analysis was performed by capillary electrophoresis (CE-UV) in the micellar electrokinetic chromatography mode. The authors stated that by using their on-line coupled SDME technique, the detection sensitivity could be increased by a factor of 1583 up to 3556. On-line extraction in screening analyses On-line SPE

An overview of the methods using on-line SPE covered in this article can be seen in Table 5. In 2006, Schönberg et al. described a screening method that was capable of detecting approximately

Table 4. Single-analyte or multicomponent methods using single-drop microextraction. Year

Analytes

Sample Extraction Validation Analytical Advantages volume and system technique matrix

2011

Berberine, palmatine and tetrahydropalmatine

4 ml urine

n-octanol/ Proof of water principle

CE-UV

Disadvantages

High concentration No validation factors achievable and high sample demand  [29]

CE: Capillary electrophoresis.

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∼2600

42

365

Six model compounds of basic drugs

2006

2010

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2010

2002

RAM with diol groups RAM (outer surface) and sulfonic acid groups (inner surface)

RP

RP

RP

Sorbent type

Analytical technique

Proof of principle

Yes (matrix effects, process efficiency and LOD)

Yes (matrix effects, LOD, LOQ, imprecision, imprecision of retention times and recovery)

HPLC-UV

LC–MS

UHPLC–MS

Yes (selectivity, specificity, HPLC-DAD recovery, precision, linearity, LOD, carryover, batch-to-batch reproducibility and stability)

Validation

HPLC-DAD: HPLC-diode array detection; RAM: Restricted access material; RP: reversed phase material; SPE: Solid-phase extraction.

1 ml plasma

1 ml serum HySphere™ Resin GP or urine

50 μl urine Strata X-CW

Strata® X-CW

Sample SPE sorbent volume and matrix

Number of analytes included

Year

Table 5. Screening methods using on-line solid-phase extraction.

–

Reasonable validation (except carry over) and compable to preexisting methods

Reasonable validation (except carry-over)

Reasonable validation and comparable with GC–MS

Advantages

No validation [57]

Carry-over not validated [56]

Manual hydrolysis of glucuronides and carry-over not validated [55]

Manual hydrolysis of glucuronides necessary and method includes only basic compounds [54]

Disadvantages

The current role of on-line extraction approaches in clinical & forensic toxicology

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Review  Mueller 2600 compounds using high-performance liquid chromatography-diode array detection (HPLCDAD) [54] . Sample preparation was very straightforward: after the addition of the internal standard and centrifugation, direct injection of the sample was possible for most compounds. Enzymatic hydrolysis of glucuronides was only necessary for substances with very low concentrations (e.g., psilocin). The total chromatographic run time of the method was 40 min. For screening methods, there are no generally accepted guidelines for validation at present. However, the method was reasonably validated. The authors stated that the method was compared with a commercial system (REMEDi HS from BioRad Hercules, CA, USA) and with GC–MS using 350 authentic samples and that the methods were comparable. The main drawback of the method was the selectivity of the extraction: only basic compounds were retained, while acidic and neutral substances were not retained. Substances with two basic atoms (e.g., clozapine) were retained very strongly, which would have resulted in retention times >4 h according to the authors. In 2010, Chiuminatto  et al. described a method for the simultaneous determination of 42 drugs in urine [55] . The intended goal of the method was to shorten the long analysis time for the confirmation tests of these substances in forensic toxicology. The amount of validation seemed fair. As with all screening methods, matrix effects could not be completely eliminated for all of the analytes. According to the authors, carry-over was never observed in the real patient samples they analyzed; however, no dedicated validation step was performed to check for carry-over. As with other screening methods, glucuronides needed to be cleaved enzymatically before analysis, a step that had to be carried out manually. In 2010, Sturm et al. published a screening method intended for systematic toxicological analysis in clin-

ical toxicology using on-line SPE [56] . The method was less extensively validated, but in our opinion still reasonably so, and compared with established procedures using 12 serum and 13 urine samples. The authors concluded that the main advantage of their method was the fast processing of serum and urine samples sent to their laboratory for toxicological analysis. In 2002, Chiap et al. published a paper studying the application of a then new type of online SPE sorbent, a RAM sorbent coated with diol groups on the outer surface and sulfonic acid groups on the inside [57] . This sorbent was also compared with another already-known reversed-phase-type sorbent. The paper described the optimization of the extraction method for six model compounds of hydrophilic, basic drugs. The authors established generic conditions, therefore potentially enabling the use of this extraction technique for broader screening applications. The authors concluded that the new type of RAM was superior to the previously used RAM ­sorbent. Turbulent flow chromatography

An overview of the methods using TFC covered in this article can be seen in Table 6. Mueller et al. published a paper describing the development and validation of a screening method using TFC as an on-line extraction technique [22] . The method was reasonably validated. Compared with similar screening methods using off-line extraction, the method used only a small sample volume; nevertheless, limits of identification were at least comparable, if not better. Samples were analyzed twice, once natively and once after enzymatic hydrolysis, which prolonged the total turnaround time, but increased the detectability of extensively glucuronidated substances. Besides enzymatic hydrolysis of glucuronides, the addition of internal standards and buffer plus centrifugation,

Table 6. Screening methods using turbulent flow chromatography. Year

Number of analytes

Sample TFC column Validation volume and matrix

Analytical Advantages technique

2010

356

600 μl urine Cyclone + C18XL

Yes (specificity, matrix LC–MS effects, recovery, limits of identification and reproducibility)

Small sample demand for screening method and comparable with GC–MS

Manual hydrolysis of glucuronides [22]

2012

453

100 μl Cyclone + serum or C18XL heparinized plasma

Yes (specificity, matrix effects, recovery and limits of identification)

Small sample demand and good sensitivity

Manual protein precipitation step  [58]

LC–MS

Disadvantages

TFC: Turbulent flow chromatography.

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The current role of on-line extraction approaches in clinical & forensic toxicology

no other manual steps were necessary. The method was compared with a pre-existing GC–MS method, and the results were found to be in good agreement. The sample turnaround time with the TFC–LC–MS method was substantially faster than with the GC–MS method. The same method was validated for serum and heparinized plasma [58] , and the obtained results were compared with clinical data. For approximately 80% of all substances, the limits of identification were below the minimal therapeutic concentrations using only 100 μl of serum or heparinized plasma, which illustrates the sensitivity that is achievable for a broad screening method using on-line extraction. However, with regards to manual sample pretreatment, besides the addition of the internal standard and centrifugation, a PPT step was necessary. Conclusion Currently, usage of on-line extraction in clinical and forensic toxicology is relatively scarce, but it is increasing. The published methods cover a wide range of substances and matrices, proving the applicability of on-line extraction in clinical and forensic toxicology. The published methods also prove that a significant simplification of sample preparation for routine work is achievable with on-line extraction. However, today, most methods still need some manual steps (e.g., PPT of whole blood samples with subsequent centrifugation). At present, most methods published in the literature are methods for the identification and/or quantification of single compounds or multicomponent methods. To the best of our knowledge, only a few methods usable for systematic toxicological analysis or general unknown screening have been currently published in the literature. However, these papers prove that on-line extraction is also a beneficial tool for screen-

Review

ing approaches, simplifying sample preparation and ­shortening ­turnaround times. Future perspective The use of on-line extraction will almost certainly increase in the future, because in clinical and forensic toxicology, turnaround times, throughput of samples and cost will become increasingly important. Besides more extensive usage of on-line extraction in its various forms, a more extensive use of automation is also very likely to occur. Today, even with the use of online extraction, manual sample preparation steps are necessary (e.g., centrifugation, PPT [for whole blood samples] or hydrolysis of glucuronides). Further developments might be similar to those seen in clinical chemistry with its fully integrated random-access analyzers, where every step after loading of the sample onto the analyzer is fully automated. Another active field of research is miniaturization, which involves achieving higher sensitivity with the usage of a reduced sample volume. On-line extraction is a versatile technique for miniaturization, as sample loss during the extraction procedure is minimized. Supplementary data To view the supplementary data that accompany this paper please visit the journal website at: www.future-science.com/doi/full/10.4155/BIO.14.179

Financial & competing interests disclosure The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

Executive summary Background • Automation is becoming more and more important in the clinical and forensic toxicological laboratory. • Extraction is usually the most time-consuming step; therefore, it is reasonable to automate this step.

On-line extraction techniques used in clinical & forensic toxicology • Various on-line extraction techniques are currently used in clinical and forensic toxicology, such as on-line solid-phase extraction, turbulent flow chromatography, solid-phase microextraction, microextraction by packed sorbent, single-drop microextraction and on-line desorption of dried blood spots. • Various publications describing quantitative and qualitative methods for the analysis of toxicologically relevant chemicals, including methods that are suitable for systematic toxicological analysis, prove the applicability of on-line extraction techniques in clinical and forensic toxicology.

Conclusion & future perspective • Usage of on-line extraction in clinical and forensic toxicology will almost certainly increase in the coming years.

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