Applications of Liquid Chromatography- Mass Spectrometry ......Liquid chromatography-mass...

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Journal of Analytical Toxicology, Vol. 21, March/April 1997 Applicationsof Liquid Chromatography- Mass Spectrometryin Analytical Toxicology: A Review Haraid Hoja 1, Pierre Marquet 1,', Bernard Verneuil 1, Hayat Loffi 1,z, Bernard P~nicaut 1, and G~rard tacb~tre 1,~ I Department of Pharmacology and Toxicology, University Hospital, 87042 Limoges, France and 2Laboratory of Toxicology, Faculty of Pharmacy, 87025 Limoges, France Abstract Liquid chromatography-mass spectrometry (LC-MS), after long- term development that has introduced seven major interfacing techniques, is finally suitable for application in the field of analytical toxicology. Various compound classes can be analyzed, and sensitivities for more or less polar analytes that are as good as or better than those of gas chromatography-mass spectrometry can be obtained with modern interfaces. In addition, because ionization is often softer than classical electron impact, some LC-MS interfaces are able to handle fragile species that are otherwise not amenable to MS. This review is intended to present LC-MS to lessfamiliarized readers and to give an extensive overview of the application of the different coupling techniques to doping agents, drugs of abuse, forensic analysis, toxic compounds of various nature, and several toxicologically relevant therapeutic drugs. Experimental parameters such as the interfaces used, ionization methods, detection limits, and experimental details for exemplary applications are given. Introduction The coupling of mass spectrometry (MS) as a detector to chromatographic separation systems may be the response to identification and quantitation problems often encountered by analytical toxicologists. If the sample matrix is complex (plasma, urine), multiple extraction steps are usually required before analysis. In spite of the quality of purification, interfer- ences that make proper identification and quantitation im- possible may occur even with detectors such as ultraviolet, fluorimetric, and electrochemical detectors with liquid chro- matography (LC) and electron capture and nitrogen-phos- phorus detectors with gas chromatography (GC). MS detectors using electron impact (EI) ionization operated in the scan mode can provide nearly unambiguous spectral information about compound identity, and, when operated in the selected * Address to which correspondence should be sent:Dr. P. Marquet,Service de Pharmacologie et Toxicologie,CHRU Oupuytren, 2 Avenuedu Pasteur Martin-Luther-King,87042 Limoges Cedex,France. ion monitoring (SIM) mode, can ally excellent sensitivity with high specificity. In GC, the interfacing problems with MS were overcome a long time ago. Reliable instruments that are currently part of the standard equipment of the modern toxicological laboratory are available. It is well-known that nearly 70% of everyday samples being treated in toxicological laboratories can be handled by LC. Unfortunately, the situation for LC coupling is different because LC-MS instruments are far more expensive than their GC counterparts, which partially explains why they are used less frequently. Various interfaces have been constructed since the early 1970s. Each interface applies a different technique to eliminate the chromatographic solvent, which, if introduced directly to the high-vacuum region of a mass spectrometer (10 -5 Tort), would supply an additional gas load of up to several hundred liters per rain. Sensitivity of LC-MS depends both on the compound and on the interface used. In a way, LC-MS can be seen as a straightforward approach for the analysis of polar or thermolabile compounds because no derivatization step is required as in GC-MS. Heat, if applied, is hardly sufficient for analyte degradation. It has also been shown that rapid heating does not necessarily lead to compound decomposition because the kinetics for the competing decomposition and ionization processes are often in favor of the latter (1). The ability of some LC-MS techniques to assess intact drug conjugates (i.e., glutathione, glucurono-, and sulfo-conjugates), which is impossible in GC-MS, greatly facilitates identification and quantitation of such compounds, avoiding hydrolysis steps often applied in analytical toxicology. Some LC-MS interfaces are even suitable for the analysis of peptides and proteins, some of which are drugs, some of which are toxic. The aims of the present review are as follows: to clarify the ideas of the potential user concerning LC-MS interfaces, their powers, and weaknesses and to comprehensively review the literature of its application in analytical toxicology. Description of ionization methods and LC-MS interfaces It is beyond the scope of this article to 'give an extensive overview of the processes involved in analyte ionization. There- 1 1 6 Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.

Transcript of Applications of Liquid Chromatography- Mass Spectrometry ......Liquid chromatography-mass...

Page 1: Applications of Liquid Chromatography- Mass Spectrometry ......Liquid chromatography-mass spectrometry (LC-MS), after long- term development that has introduced seven major interfacing

Journal of Analytical Toxicology, Vol. 21, March/April 1997

Applications of Liquid Chromatography- Mass Spectrometry in Analytical Toxicology: A Review Haraid Hoja 1, Pierre Marquet 1,', Bernard Verneuil 1, Hayat Loffi 1,z, Bernard P~nicaut 1, and G~rard tacb~tre 1,~ I Department of Pharmacology and Toxicology, University Hospital, 87042 Limoges, France and 2Laboratory of Toxicology, Faculty of Pharmacy, 87025 Limoges, France

Abstract

Liquid chromatography-mass spectrometry (LC-MS), after long- term development that has introduced seven major interfacing techniques, is finally suitable for application in the field of analytical toxicology. Various compound classes can be analyzed, and sensitivities for more or less polar analytes that are as good as or better than those of gas chromatography-mass spectrometry can be obtained with modern interfaces. In addition, because ionization is often softer than classical electron impact, some LC-MS interfaces are able to handle fragile species that are otherwise not amenable to MS. This review is intended to present LC-MS to less familiarized readers and to give an extensive overview of the application of the different coupling techniques to doping agents, drugs of abuse, forensic analysis, toxic compounds of various nature, and several toxicologically relevant therapeutic drugs. Experimental parameters such as the interfaces used, ionization methods, detection limits, and experimental details for exemplary applications are given.

Introduction

The coupling of mass spectrometry (MS) as a detector to chromatographic separation systems may be the response to identification and quantitation problems often encountered by analytical toxicologists. If the sample matrix is complex (plasma, urine), multiple extraction steps are usually required before analysis. In spite of the quality of purification, interfer- ences that make proper identification and quantitation im- possible may occur even with detectors such as ultraviolet, fluorimetric, and electrochemical detectors with liquid chro- matography (LC) and electron capture and nitrogen-phos- phorus detectors with gas chromatography (GC). MS detectors using electron impact (EI) ionization operated in the scan mode can provide nearly unambiguous spectral information about compound identity, and, when operated in the selected

* Address to which correspondence should be sent: Dr. P. Marquet, Service de Pharmacologie et Toxicologie, CHRU Oupuytren, 2 Avenue du Pasteur Martin-Luther-King, 87042 Limoges Cedex, France.

ion monitoring (SIM) mode, can ally excellent sensitivity with high specificity.

In GC, the interfacing problems with MS were overcome a long time ago. Reliable instruments that are currently part of the standard equipment of the modern toxicological laboratory are available.

It is well-known that nearly 70% of everyday samples being treated in toxicological laboratories can be handled by LC. Unfortunately, the situation for LC coupling is different because LC-MS instruments are far more expensive than their GC counterparts, which partially explains why they are used less frequently. Various interfaces have been constructed since the early 1970s. Each interface applies a different technique to eliminate the chromatographic solvent, which, if introduced directly to the high-vacuum region of a mass spectrometer (10 -5 Tort), would supply an additional gas load of up to several hundred liters per rain. Sensitivity of LC-MS depends both on the compound and on the interface used. In a way, LC-MS can be seen as a straightforward approach for the analysis of polar or thermolabile compounds because no derivatization step is required as in GC-MS. Heat, if applied, is hardly sufficient for analyte degradation. It has also been shown that rapid heating does not necessarily lead to compound decomposition because the kinetics for the competing decomposition and ionization processes are often in favor of the latter (1). The ability of some LC-MS techniques to assess intact drug conjugates (i.e., glutathione, glucurono-, and sulfo-conjugates), which is impossible in GC-MS, greatly facilitates identification and quantitation of such compounds, avoiding hydrolysis steps often applied in analytical toxicology. Some LC-MS interfaces are even suitable for the analysis of peptides and proteins, some of which are drugs, some of which are toxic.

The aims of the present review are as follows: to clarify the ideas of the potential user concerning LC-MS interfaces, their powers, and weaknesses and to comprehensively review the literature of its application in analytical toxicology.

Description of ionization methods and LC-MS interfaces It is beyond the scope of this article to 'give an extensive

overview of the processes involved in analyte ionization. There-

1 1 6 Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.

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fore, the principle of each method used in LC coupling will briefly be cited, and more detailed information may be found elsewhere (2). A succinct description of the interfaces will also be made and more profound information may be found cited in the literature. The different types of mass analysis, however, will be presented in a more detailed manner because the ion- ization methods used in LC-MS and the chemical background noise of the solvent in the low-mass region sometimes make the use of tandem mass spectrometers (MS-MS) necessary.

Ionization occurs upon bombardment with electrons (EI), upon exposure to primary ions at medium or atmospheric pressure (chemical ionization, CI), upon bombardment with accelerated atoms (fast-atom bombardment, FAB) or ions (sec- ondary ion mass spectrometry, SIMS), or upon desorption of ions from charged droplets obtained by thermospray (TS) or electrospray (ES) nebulization.

The desorption methods like ES, TS, and FAB and atmospheric-pressure chemical ionization (APCI) are "soft" ionization modes, which means that, in favorable cases, fragile species (i.e., thermolabile molecules and some drug-conju- gates) can be transformed into ions without decomposition by these techniques. In addition, ES offers the possibility of producing multiply charged ions, which is interesting if biomolecules are assessed.

Moving belt (MB) The LC flow (1-2 mL/min) is deposited on a belt that trans-

ports a sample successively through a heated desolvation chamber and a two-stage differentially pumped region into the low-pressure ion source. Ionization may be obtained by several complementary ionization modes: EI, CI, SIMS, or FAB. If the identification of unknown compounds is desired, EI is used. For highly sensitive analyses, CI is preferable, whereas for biomolecules, FAB is the method of choice. The power of MB lies in its ability to accommodate EI as well as other types of ionization. Its inconveniences are belt memory effects when handling highly aqueous solvents and discrimination or decomposition of low-volatility samples by application of heat during the desolvation step (3-7).

Particle beam (PB) The chromatographic solvent (0.6-2 mL, depending on its

volatility) is nebulized at atmospheric pressure into a slightly heated desolvatation chamber and is transferred into the low- pressure ion source through a two-stage jet separator. Ioniza- tion is obtained by EI, CI, or FAB. PB has the advantage of accommodating EI, whereas its major weakness is the pos- sible discrimination of low-volatile samples in the jet-sepa- rating region (8,9).

Direct liquid introduction (DLI) Direct liquid introduction relies on the direct nebulization of

the chromatographic effluent into the source of the mass spec- trometer where solvent-induced CI occurs. The solvent is evac- uated by the vacuum pumps. The highest solvent flow rate usable with MS is approximately 50 laL/min; therefore, the tech- nique needs either microbore LC or postcolumn splitting of the chromatographic flow. Good sensitivities can be obtained, but

solvent splitting yields a concomitant loss of sensitivity due to the mass-flow sensitivity of MS detectors (10-12).

Continuous-flow FAB (CF-FAB), frit-FAB, and SIMS A glycerol-containing solvent (5 laL/min) is introduced with

an open-ended (CF-FAB) or frit-terminated (frit-FAB) capillary into the low-pressure ion source of the mass spectrometer, where it is bombarded with accelerated atoms. The ions formed are extracted by an electrode and transferred to the analyzer. The disadvantages of this technique are background-ion con- tributions of the matrix, which can interfere occasionally, and very limited solvent flow rates, which impose a solvent split before its introduction to the instrument.

SIMS can be performed with the same type of interface when using ions instead of atoms for analyte bombardment (13-15).

Thermospray The solvent (up to 2 mL/min) passes through a heated

capillary in which it is almost completely volatilized. Then it is nebulized into an expansion chamber where ionization takes place at low pressure, induced by the solvent buffer, by a fila- ment, or by a discharge electrode. After complete desolvation, the analyte ions are transferred to MS by means of a repeller electrode. Ionization corresponds to solvent-induced CI in the three modes that differ only with respect to intensity and to fragmentation pattern. Fragmentation can also be enhanced by applying higher voltages to the repeller electrode and acceler- ating the desolvated ions, which can give fragmentation upon collision with residual solvent molecules (16-19).

Electrospray A flow rate of 50 laL/min of solvent is nebulized both by

high voltage applied to a silica capillary and by pneumatic assistance. The ionization process takes place under atmo- spheric pressure in the liquid phase. After desolvation, ion- solvent molecule clusters are destroyed through acceleration in the transition region between atmospheric pressure and vacuum and assisted in some instruments by a countercurrent stream of nitrogen. The acceleration of ions in this transition region may be sufficiently energetic to induce fragmentation upon ion-molecule collisions. The need for postcolumn splitting does not decrease sensitivity because the response of the system is concentration-sensitive and not mass-flow- sensitive (20-23).

Atmospheric-pressure chemical ionization Solvent nebulization (up to 2 mL/min) is performed at

atmospheric pressure by a heated nebulizer with pneumatic assistance. A discharge electrode ionizes solvent molecules, which, after several ion molecule reactions, transfer a charge to the analyte molecules. Fragmentation can be induced in the same manner as for ES (24).

Mass Analysis

The ions obtained undergo mass analysis in appropriate mass filters. Quadrupole mass filters are used most frequently,

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ion traps were implemented recently, and sector instruments can be accommodated by some interfaces. In general, if high mass resolution is not necessary, quadrupoles are preferred because of their ruggedness. The filters can be operated by scanning a specified mass range (scan mode) or by scanning selected masses (SIM). A more than 10-fold increase in sensi- tivity is obtained in SIM experiments compared with full scanning. Coupling of two mass analyzers (MS-MS) gives additional operation features, which are particularly valuable for the identification of unknowns or for screening purposes. An inert target gas, which is introduced between the two mass filters, enhances fragmentation. This operation is called colli- sion-induced dissociation (CID).

Four types of experiments are possible: �9 Daughter-ion scanning: A parent-ion mass is selected in the

first mass filter and CID fragments are collected by scan- ning the second one. Identification of unknowns is easier because the ions observed can only be issued from the parent ion selected.

�9 Parent-ion scanning: A daughter-ion mass is fixed in the second mass filter while the first one is scanned. Ions giving fragments at masses corresponding to the one selected in the second mass filter give a signal, whereas others do not. In this way, parent ions or fragmentation pathways can be identified easily.

�9 Constant neutral-loss scanning: The two mass filters are scanned by maintaining a given mass difference which corresponds to the loss of neutral species from the parent ion. This operation is used when scanning for specific drug conjugates.

�9 Selected-reaction monitoring (SRM), the MS-MS equiva- lent of SIM: Only ions which correspond to a specific frag- mentation route are monitored. This type of experiment enhances selectivity and sensitivity of analysis in the same manner as SIM in a single analyzer mass spectrometer.

Toxicological Applications Criteria for choosing the articles were as follows: the articles

dealt with 1. applications of LC-MS to drugs and toxic com- pounds that were found in human or animal biological fluids or in biological matrices that are normally absorbed by humans and 2. a few comparisons of two or more interfaces for the assessment of toxicologically relevant drugs or toxins which were included even if pure compounds were studied.

Doping agents and drugs of abuse This section comprises steroids, anabolics, diuretic drugs,

carbamate doping agents, nonsteroidal anti-inflammatory drugs (NSAIDs), opiates, and analgesics. Applications with doping agents in biological fluids of animals were included because the extraction procedures remain the same regardless of the species and can easily be adapted for the assessment of these compounds in biological fluids of humans.

Steroids. MB was used to quantitate progesterone in human serum with CI, SIM detection, and deuterated progesterone as

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internal standard. The detection limit was rather high (4 ng/mL or 200 pg injected) (25). PB, also with CI and SIM detection, allowed the quantitation of beclomethasone and its derivatives in human serum with a quantitation limit for long-time routine use voluntarily fixed at 1 ng/mL (120 pg injected) (26).

ES and APCI were applied for methandrostenolone and its metabolites in equine urine. APCI was more suitable for neu- tral compounds, and ES, which works best for compounds that are ionic in solution, was used for the sulfo-conjugated metabolite. As both techniques gave few fragment ions, frag- mentation had to be induced by CID (27). Boidenone and five related steroid sulfo-conjugates were assessed in equine urine by negative ionization ES coupled to MS-MS. Using simple mass analysis only, detection limits were 3--4 ng and 100-350 pg injected for full scan and SIM of the molecular ion, respec- tively. With MS-MS, the selectivity for confirmation and quan- titation increased. Full-scan daughter ions could be obtained for 5-10 ng injected, and SRM of two fragment ions gave detection limits of 110-370 pg oncolumn. With this experi- mental setup, the sulfo-conjugate of boldenone could be detected up to 17 days postdose (28).

Other authors, who used TS for confirmation of a number of corticosteroids in equine urine, carefully optimized solvent composition to enhance ionization efficiency. Approximately 10 ng of prednisone or dexamethasone injected was sufficient for a full-scan thermospray spectrum in the positive ion mode. Negative ionization was slightly more sensitive but lacked frag- mentation (29). The aforementioned results are consistent with those obtained for the assessment of 10 corticosteroids in human urine by TS. Detection limits ranged from 10 to 50 ng injected for full scan spectra and ranged from 1 to 5 ng with SIM (3O).

Other applications used DLI for several corticosteroids (31,32), betamethasone (33,34), and diethylstilbestrol (34) and used TS (35) and APCI (36) for trenbolone.

~-agonists. [3-agonists were assayed by TS, ES, and APCI. When using SIM detection, clenbuterol could be assessed in human plasma at a detection limit of 0.1 ng on-column by APCI (37). Comparing GC-MS, TS, and ES for the determina- tion of five ~-agonists, an equivalent sensitivity for GC-MS and ES (100 pg and I ng by SIM and scan mode, respectively) was obtained. Both methods gave 50-fold lower detection limits than TS (38).

Diuretics. DLI and TS were used for the determination of diuretic drugs. The DLI application attained a detection limit of approximately 1 ng injected for trichlormethiazide on a laboratory-built device with microbore LC as a separation system (39). TS was used for confirmation of 12 diuretics and the uricosuric probenecide in human urine, and nine were identified in real-life extracts. Use of gradient elution with TS lead to instability in ionization efficiency, therefore three iso- cratic systems were applied. Scans were obtained for 50 ng injected for all but three compounds, which was sufficiently low for valuable confirmation (40). Benzthiazide, a diuretic that was not amenable to GC-MS by routine derivatization methods, could be confirmed by TS after optimization of the LC solvent in order to enhance ionization (41).

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Carbarnate. A screening procedure for urinary metabolites of mesocarb, which is a nontherapeutic doping agent of the car- bamate family, used PB with EI for confirmation of the intake of the parent drug. An LC-MS method was necessary as the drug was not amenable to GC (42).

NSAIDs. PB using three ionization modes and ES were com- pared as confirmational devices for flunixine in equine urine. The detection limit for PB was 10 ng/mL, whereas ES was more sensitive (value not given) but needed CID to enhance fragmentation. A list of 40 NSAIDs giving the intensity of the response for the three PB ionization modes and the three most abundant ions was included (43).

Drugs of abuse. TS was found suitable for the quantitation of morphine and codeine in human urine in a study on standard reference material. The results obtained were in good agree- ment with GC-MS and direct-probe MS-MS. The detection limit for TS was estimated to be 2 ng/mL (44). Other drugs of abuse, including methadone, dextropropoxyphene, and dextro- moramide, gave excellent sensitivities in whole blood with TS and SRM. Detection limits were 10-100 pg for MS-MS (45). LSD was detected in urine with a detection limit of 200 pg for DLI with SIM detection using a coupled-column technique and heart-cutting of the peak of interest (46). Other applications used CF-FAB for dextromethorphan with a determination limit of 110 ng/mL, whereas the plasma concentrations could be as low as 10 ng/mL (47) and TS for methamphetamine (48).

Identification of unknowns and screening procedures in forensic analysis

MS coupling becomes particularly valuable when unknown compounds are to be identified, provided that the spectrum gives enough informative fragments for interpretation. The applications found on the subject use DLI, MB, PB, frit-FAB, TS, ES, and APCI. Use of EI spectra is advantageous because of the possibility for library searching, although molecular weight information may be absent. This is not a major drawback because the interfaces using EI are all able to handle CI, which most often provides molecular weight information. In this case, quantitation is performed with CI and SIM detection because spectra are simpler, which gives less rise to interfer- ence from other compounds.

Using MB with EI and positive CI allowed the identification of unknown compounds, which were found during a screening procedure for benzodiazepines in postmortem plasma and urine, as the pesticide diuron and four of its metabolites. Con- firmation of compound identity was realized by high resolution measurements on a sector instrument in order to measure the exact molecular weight. The detection limit was 50-80 ng for a scan. LC-MS was, in this case, superior to GC-MS because the compound was thermolabile (49,50). The same group used MB with CI for the quantitation of clopenthixol, bromazepam, and reserpine in serum taken from an individual who attempted suicide. Detection limits by SIM ranged from 35 to 100 pg. The presence of the compounds in serum, except that of reserpine whose concentration was too low, could be confirmed with accurate mass measurements with a sector instrument (51).

Electrospray MS-MS was used to identify and to quantitate

a toxic contamination in a dexamethasone-injectable solution. Analysis was quite demanding because the compound suc- cinylcholine could not be separated from the adjuvants of the drug formulation because of its hydrophilic nature. For this reason, it was overlooked in routine screening procedures (52).

A routine APCI screening procedure for 21 organophosphor- ous pesticides in blood was developed for cases of intoxication by agrochemicals. After optimizing mobile phase composition to enhance sensitivity, detection limits ranged from 200 to 1000 ng for scanning and from 2 to 50 ng for SIM, which was only slightly inferior to GC-MS. Positive or negative ionization was used, depending on the compound, and two or three con- firmatory fragment ions could be obtained for each compound (53).

The pesticide ethylenethiourea was detected in urine down to 0.1 ng injected with TS and SIM of the protonated molecular ion (54). A thermospray screening method for 21 benzodi- azepines used the combined information of retention times and isotopic ratios, which are related to the presence of halogen, to identify positive samples. Although peaks were not completely resolved when acquiring the total ion current (TIC), their res- olution could be obtained with the selected ion channels (55).

Frit-FAB with micro-LC using an online concentration column was applied to several forensic applications. Full scans of benzodiazepines, cephalosporine antibiotics, and phenothi- azines could be obtained with detection limits of 4-10 ng/mL, 10-100 ng, and 0.25-10 ng on column, respectively (56).

A DLI source was used to detect metabolites of the explosive 2,4,6-trinitrotoluene in human urine with an SIM detection limit of 0.1 ng/mL (57), and explosives and their mixtures could be identified in 1 to 10 I~g of sample (58).

ES was used for penicillins and cephems in serum with a detection limit of 2-15 ng for scanning (59). TS was used for thermolabile benzodiazepines in whole blood with on column detection limits of 10-200 pg for MS-MS and SRM (60), for cyclosporins in whole blood (61), and for trimethyllead in urine (62).

Pesticides and toxins in nonhuman samples This section deals with the assessment of compounds such

as mycotoxins, marine toxins, and pesticides mostly in animal and vegetable samples. The interfaces used were MB, PB, CF- FAB, ES, and TS.

A TS method was applied for the detection of three myco- toxins and some of their metabolites, including a glucuronide conjugate, in excretion samples from rats, hens, and cows. Pos- itive filament-on ionization was preferred to negative ionization because of a more abundant fragmentation pattern. Detection limits ranged from less than 1 ng to 20 ng and from 50 to 1000 pg by scan and SIM detection, respectively (63). In another study using TS, seven mycotoxins were determined in wheat samples. Fragmentation could be enhanced or suppressed when varying the buffer concentration. Very low concentrations and filament-enhanced ionization yielded fragments for three of the compounds, whereas higher concentrations favored the protonated molecular ion and lowered detection limits. Overall detection limits by SIM were 75-1000 pg, and calibration was linear in the range of 3-40 ppb (ng/g) (64).

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Marine toxins cause severe cases of intoxication that can be lethal for fragile subjects. Domoic acid, a neurotoxic amino acid, was confirmed in shellfish with a laboratory-modified ES using SIM detection of three characteristic ions down to 0.1 tJg/g. An optimized extraction procedure from rat plasma and feces gave detection limits in the low picogram range (65). CF-FAB and SIM detection in conjunction with an open-bore fused-silica column were used for the detection of saxitoxin in human urine. ES, which was also tested by the authors, gave a 20-fold lower detection limit (66).

Confirmation of the presence of 17 organophosphorous pesticides and metabolites in beef muscle could be obtained by TS discharge-on negative ionization and SIM detection. All but four could be detected at the i ppm level or lower. Identi- fication was achieved by monitoring of the five most abundant ions. In order to increase selectivity, the retention times and the ion intensity ratios were compared with those of standard compounds (67). A number of compounds, including antibi- otics, were evaluated for their suitability for PB with EI and CI and for TS. PB with CI gave detection limits of 100 ng for al- most all of the compounds, whereas TS detection limits ranged from 10 to 400 ng for full-scan mass spectra. The interfaces were also compared for their sensitivity and specificity for the determination of cephalosporins in milk and methylene blue in muscle extracts. PB with CI was preferred to TS because SIM detection of five ions was possible with PB and CI (68). APCI, ES, TS, and PB were used for carbamate pesticides in pepper. APCI gave excellent detection limits; ES and TS performed well. PB was not sensitive enough (69).

Other applications used MB for pesticides and alkaloids (70). ES was used for the shellfish toxins okadoic acid and dinoph- ysistoxin-1 (71) and for penicillins in bovine milk (72,73), and CF-FAB was used for tetracycline antibiotics in honey (74).

Therapeutic drugs In this section, the assessment of drugs in biological fluids will

be discussed. In applications dealing with the identification and quantitation of known compounds, the use of SIM or SRM will often be encountered. LC-MS methods are often used during the early stages of drug development because they are able to provide reliable pharmacological data with minimal method-develop- ment time (75). As most of the analyses have been performed routinely for large sample series, deuterated internal standards were used frequently in order to minimize intra-assay varia- tions. For studies in which the elucidation of metabolites was de- sired, MS-MS was used almost systematically.

Drugs for human usage. In a study on the suitability of APCI, various compound classes were investigated by flow injection analysis (FIA). As an example of the sensitivity of the interface, serum containing theophylline at the level of its detection limit (5 pg) was injected on column. Compound classes and the minimal detectable quantity obtained with APCI and SIM detection were as follows: amines, free amino acids, phenylhydantoin amino acids, steroids, vitamins, alkaloids (10-100 pg), peptides with molecular weights greater than 1000 Da (100 pg-200 ng), nucleosides and nucleotides (100 pg-50 ng), bile acids (10 ng), and sugars and lipids (50-200 ng) (76).

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TS was used for the assay of compounds of neurochemical interest comprising 19 indolic acids, tricyclic antidepressants, and catecholamines. The indolic compounds gave a detection limit of 50-100 pg by FIA and SIM. In addition, extraction from urine was performed successfully for five of them. Detection limits were 10 pg for the tricyclic antidepressants imipramine and clomipramine. For the previously mentioned compound classes, sensitivity, except for catecholamines, was better with TS than with GC-MS, and no derivatization step was needed for the LC method. The authors observed that for concentrations near the detection limit, additional sample cleanup would be necessary because coeluting endogenous com- pounds compete for ionization and are able to mask, to some extent, the ionization of the analyte (77). Butyrophenone tran- quilizers were quantitated in whole blood with TS and MS-MS. The SRM detection limit for haloperidol was approximately 250 pg/mL (45). The antiasthmatic drugs bambuterol, terbutaline, and budesonide were assayed by TS and SIM detection using deuterated internal standards, which gave excellent linearity of calibration graphs for gradient elutions. The quantitation limit was 0.1 ng/mL for plasma extracts of the corticosteroid budesonide (78). TS positive ionization was used to measure the enantiomers of terbutaline, a [~-stimulant with only one pharmacologically active enantiomer, in human plasma using the deuterated analogue as an internal standard. Quantitation was performed on the racemic mixture, whereas the enantiomeric ratio was determined using a coupled column technique with a chiral column. Because no chiral separation was performed on the first column, heart cutting of the peak of the racemates provided information about the enantiomeric ratio at each moment, regardless of the width of the fraction. Using this instrumental setup, linear calibration was obtained down to 4 pmol (approximately 1 rig) (79). The quaternary ammonium steroids pancuronium and vecuronium were determined in plasma and urine using MB coupled to a sector instrument (80). Other authors used ES for the sensitive mea- surement of steroids and their glucuronides in urine and reached a detection limit of 15 pg, which was better than GC-MS (81). Sumatriptan, a drug used for the treatment of migraine headaches, was determined in plasma by TS, positive ionization, and SIM. The use of a homologue of the drug as an internal standard led to high intra- and interassay variations; therefore, a labeled compound was preferred, which signifi- cantly improved results for linearity, precision, and accuracy. The routine quantitation limit was set at 2 ng/mL, although calibration graphs were linear down to 500 pg/mL (75). The same group assessed the antihypertensive drug labetalol in plasma by TS positive ionization and SIM detection. With a deuterated internal standard, the detection limit was 5 ng/mL, and the linearity ranged from 10 to 100 ng/mL (82). For phar- macokinetic studies, abanoquil, a low-dosage %-adreno- receptor antagonist, was assessed in whole blood by APCI with a deuterated internal standard. Sensitivity with MS-MS and SRM for the parent to daughter transition was excellent with a linear calibration range from 10 to 500 pg/mL (83). Anthra- cycline antibiotics used in cancer chemotherapy were investi- gated by PB with EI and CI as well as with TS in order to obtain structural information and to determine the suitability of the

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LC-MS techniques. Both LC-MS interfaces were found fit for the analysis of the drugs, but PB was preferred because it pro- vided more abundant fragment ions, though it lacked sensi- tivity compared with the other methods (84). Tacrolimus (for- merly FK-506) and some of its metabolites were determined in whole blood using PB negative CI. For quantitation purposes, the monoacetylated derivative was used as an internal standard. Other clinically relevant drugs were checked and found not to interfere with the assay. The LC-MS method was also cross- validated with an enzyme immunoassay for the drug and its metabolites in plasma, but no significant correlation could be obtained because of crossreactivity that occurred in the latter method (85). A new derivative of the fentanyl group, mirfen- tanil, which is actually under clinical investigation, was deter- mined in plasma by TS positive ionization and SIM detection. Detection and quantitation limits were 0.1 and 0.4 ng/mL, respectively, with a deuterated internal standard (86). Anti- malarial Cinchona alkaloids issued from cell cultures were assayed with TS. FIA yielded a detection limit of 10 pg for quinidine when monitoring the protonated molecular ion. The authors concluded that for the analysis of complex mixtures and accurate quantitation, the use of MS-MS and deuterated internal standards would be necessary (87). The antihistamine drug triprolidine and one of its metabolites were investigated by TS. The estimated detection limit for the parent drug was 10 pg when using SIM detection (88).

A synthetic hetrazepine derivative with platelet-activating factor (PAF) antagonizing properties was determined by PB negative ion CI with methane as a reagent gas yielding a quan- titation limit of 0.1 ng/mL (89). The performances of ES and APCI were illustrated for the detection of a PAF antagonist and a metalloproteinase inhibitor with detection limits of 100 pg/mL (90). In other applications, TS was used for the deter- mination of moricizine in plasma with a quantitation limit of 10 ng/mL (91). TS and PB were used for coumarin anticoagu- lant drugs in plasma (92), and ES was used for the pentapep- tide drug IRI-514 with a quantitation limit of 2 ng/mL (93).

Drugs for veterinary usage. A TS interface was used to confirm trimethoprim in bovine serum by SIM of the proto- nated molecular ion (94). The benzimidazole antihelmintics oxbendazole and fenbendazole were quantitated in liver and muscle of sheep using TS and SIM detection. Detection limits for oxbendazole and fenbendazole were 50 and 100 ng/g, respectively (95). Another application used APCI for medeto- midine, an analgesic sedative (96).

Metabolic studies For this section, literature was most abundant because MS

analysis can easily reveal important information with regard to metabolic pathways. For the sake of completeness and because the number of applications is important, only citations will be made.

The ionization efficiencies of APCI, ES, and TS as a function of hydrophobicity were compared for the drugs pravastatin and its metabolites and for two other drugs using positive and negative ionization. Results showed that APCI with positive ionization was the most sensitive method for nonpolar com- pounds whereas polar compounds were less suitable; the best

sensitivity with TS for hydrophobic compounds was obtained with negative ionization; and ES was very sensitive regardless of compound polarity and ionization mode and was the most sensitive interface for hydrophilic compounds (97).

Thermospray was used for the identification of an unknown urinary metabolite of heptabarbital (98), for the antiepileptic drug lamotrigine (99), for glucuronide metabolites of doxyl- amine (100), for SK&F 101468, a low-dosage dopamine-d2 re- ceptor agonist (101), for haloperidol (102), for the uricosuric drug benzbromarone (103), for nicotine and its glucuro-con- jugates (104), for metabolites of temelastine SK&F 93944 (105), for chlorpromazineoN-oxide, an antipsychotic agent of the phenothiazine family (106), for the DNA adducts of the anticancer drugs mitomycin C, porfiromycin, and thiotepa (107), for acetaminophen conjugates (108), for coumarinic anticoagulants (109), for terfenadine (110), and for ampicillin antibiotics (111).

ES was used for N-0923, a hydroxytetralin enantiomer with potent dopamine-d2 receptor agonizing activity (112); for S 12813, a drug with analgesic activity (113); for clozapine N § glucuronide (114); and for vitamin D3 metabolites (115).

CF-FAB was used for vitamin D3 metabolites (116) and for ampicillin antibiotics (111); PB with EI was used for oxodipine, a new calcium agonist (117); DLI was used for a glucurono- conjugate of flurazepam (118); and MB was used for the synthetic corticosteroid budesonide (119).

Discussion

The principle functions of the seven LC-MS interfaces which were or are commonly used have briefly been presented, and their respective advantages and disadvantages have been pointed out. The applications of these interfaces covering various fields of toxicological analysis have been extensively reviewed.

Because of their more or less polar natures, all of the compounds were well-suited liquid chromatography analytes. The applications have been grouped following previously defined toxicological fields; a discussion about the interest of the interfaces with respect to compound classes may give a clearer understanding of their respective performance.

Direct liquid introduction (DLI), with which MS coupling was realized for the first time, was rarely applied although good limits of detection could be reached for the analysis of explosives and, with an appropriate experimental setup, for LSD.

Applications with the moving belt (MB) were also rare. Its sensitivity for steroids was mediocre, whereas benzodiazepines were detected more sensitively. The major advantage of the interface, which is its potential to obtain library-searchable EI spectra, was only used in confirmational studies for rela- tively high concentrations because its sensitivity was poorer than that of CI.

Applications using particle beam (PB) were found more often. Sensitivities for steroids and NSAIDs were good, and those for antibiotics were mediocre. The instrument was not

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convenient for the analysis of pesticides. Just as with MB, EI ionization was not used when sensitivity was desired.

Fast atom bombardment was used for the analysis of ben- zodiazepines, antibiotics, and phenothiazines with very good performance because of an experimental setup that used on- line column preconcentration. However, dextromethorphan could not be quantitated in plasma using FAB because its quantitation limit was too high.

Thermospray was the interface that was applied most often and that was used in nearly all fields. Its performances were equivalent to or better than the previously discussed inter- faces. High sensitivities could be obtained for steroids, drugs of abuse, thiourea pesticides, mycotoxins, indolic compounds, and tricyclic antidepressants. Its application range included many therapeutic drugs and metabolic studies.

Electrospray is a relatively new interface that has been applied for steroids, diuretics, NSAIDs, carbamate pesticides, and marine toxins. Limits of detection were at least of the same order as those of TS. Further, ES was sometimes used for the determination of therapeutic drugs and for metabolic studies.

Atmospheric pressure chemical ionization gave low detec- tion limits for steroids, 13-agonists, carbamate pesticides, alka- loids, and vitamins. A comparison with ES and TS showed that APCI was the most sensitive interface for pesticides and 13-agonists.

This overview shows that DLI, MB, PB, and FAB have found restricted application in analytical toxicology, whereas APCI and ES were used more often and TS was used very frequently. Good sensitivities could be obtained with all of them, provided the compound chosen ionizes well. Valuable conclusions with regard to the suitability of each interface cannot be drawn if one compares only their respective number of applications because some of them have been used for nearly 20 years (MB, DLI), whereas others are comparatively new (APCI, ES).

For the three most promising interfaces, APCI, ES, and TS, a comparative study of instrument sensitivity as a function of compound polarity has shown that ES was applicable for a wide range of analyte polarity, whereas APCI and TS were better suited for hydrophobic compounds (97). These tech- niques have a relatively soft ionization in common, so the mass spectra sometimes lack fragment ions. Fragmentation can be enhanced for all three by accelerating the ions in regions of intermediate pressure where collision with neutral molecules occurs. Nevertheless, confirmational fragments are usually less abundant than with classical EI ionization. If this first method is not efficient enough, collision-induced disso- ciation in tandem mass spectrometry provides fragmentation in a more controlled manner.

With regard to the frequency of their use and bearing in mind that ES and APCI have certainly not been developed to their full potential, one can think that these two interfaces and TS are actually the most interesting techniques for toxicological anal- ysis. If genetic therapy is developed, ES could play a more important role because the analysis of proteins can easily be performed, taking advantage of its ability to form multiply charged ions that can be determined on common quadrupole mass filters. Moreover, ES, which handled the widest range of compound polarity with a good sensitivity, and APCI, which

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showed the highest sensitivity for lipophilic analytes, can be per- formed on the same instrument by simply changing the ion source. Such a combination may replace thermospray in the near future.

Conclusion

LC-MS has been shown to be applicable to samples of various natures that may have a wide range of polarity. The interfacing problem that hampered its rapid applicability seems to have been overcome in more than 20 years of development. Some of the interfaces, which either had the merit to be pioneers in the field of LC-MS or showed new perspectives when introduced, lack sensitivity when compared with more recent instruments. This is why it may be justified to classify them according to their suitability for trace analysis and their potential interest for toxicological applications.

Instruments of historical interest. Direct liquid introduction and moving belt have virtually disappeared because they are technically demanding and are not sensitive enough for trace analysis.

Instruments of secondary interest. Particle beam and con- tinuous-flow or frit fast atom bombardment have proved to be valuable tools for identification and quantitation: PB because of the library searching possibility for spectra acquired with electron impact ionization and CF-FAB because ionization is relatively soft and because it is able to assess compounds of high molecular mass. Nevertheless, these may also eventually disappear. PB lacks sensitivity when used with EI, and there are better methods available for use with CI. FAB can only achieve low detection limits with coupled column methods that are technically demanding.

Instruments of primary interest. Thermospray, electrospray, and atmospheric pressure chemical ionization are the methods that were most often applied in the past years. In the mid-1980s and the beginning of the 1990s, LC-MS coupling and TS were nearly indiscernable, as can be seen by the tremendous number of applications published using this interface. The limits of detection and the reliability of the instrument are satisfying, and variations in ionization efficiency often require the use of labeled internal standards. ES and APCI have found wider use in the last seven years. These methods, which are generally used on the same type of instrument by changing the ion source, are complementary with regard to sensitivity as a function of the polarities of the analytes. The excellent sensi- tivity attained for hydrophobic compounds by APCI, the wide range of solute polarity, and the ability to assess multiply charged ions by ES makes this coupling a highly performing instrument that should gain more importance in the future when peptide and protein drugs will be used more frequently.

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Manuscript received January 8, 1996; revision received April 26, 1996.

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