Application of gc gcms hplc and hplcms for drug analysis

Although immunoassays are widely used for routine TDM in clinical laboratories, other analytical techniques such as GC, HPLC, GC/MS, and HPLC/MS are also used for determination of concentrations of various drugs in clinical laboratories (Table 2). These alternative techniques, especially GC/MS and HPLC/MS, are very sensitive and specific for a chosen analyte whereas immunoassays suffer from multiple problems including interferences from compounds with similar structures, hook effect, and sensitivity. There is no commercially available immunoassay for analysis of antiretro-virals used in the treatment of patients with HIV infection. HPLC methods or HPLC combined with tandem mass spectrometry are the only available techniques for therapeutic monitoring of these drugs. GC, GC/MS, and HPLC are also preferred methods for TDM of tricyclic antidepressants because commercially available fluorescence polarization immunoassay (FPIA) cross-reacts with all tricyclic antidepressants and their metabolites. Although immunoassays are available for routine monitoring of certain immunosuppressant drugs, these assays suffer from many limitations and HPLC combined with tandem mass spectrometry can be used for therapeutic monitoring of these drugs (15).

6.1. Analysis of Anticonvulsants

Immunoassays are commercially available for TDM of antiepileptic drugs including phenytoin, carbamazepine, phenobarbital, ethosuximide, primidone, and valproic acid. Before development of immunoassay techniques, conventional antiepileptic drugs were analyzed by GC and HPLC in clinical laboratories. Kuperberg (16) described a GC method for quantitative analysis of phenytoin, phenobarbital, and primidone in plasma. Another GC method for simultaneous analysis of phenobarbital, primidone, and

Table 2

Application of Gas Chromatography (GC) and High-Performance Liquid Chromatography (HPLC) for Analysis of Drugs in Serum/Plasma Where There is No Commercially Available

Immunoassay

Table 2

Application of Gas Chromatography (GC) and High-Performance Liquid Chromatography (HPLC) for Analysis of Drugs in Serum/Plasma Where There is No Commercially Available

Immunoassay

Drug

Class

Analysis

Reference

Carbamazepine

Anticonvulsant

HPLC

(20)

and 10,11-epoxide

GC/MS

(21)

Gabapentene

Anticonvulsant

HPLC

(23)

Lamotrigine,

Anticonvulsant

HPLC

(24)

Oxcarbazepine, Felbamate

GC/MS

(25)

Lamotrigine

Pregabalin

Anticonvulsant

HPLC

(26)

Mexiletine

Cardioactive

HPLC

(33)

GC/MS

(34,35)

Flecainide

Cardioactive

HPLC

(37)

GC/MS

(38)

Tocainide, Verapamil

Cardioactive

HPLC

(30)

Amiodarone and other drugs

Cardioactive

Encainide

HPLC

(31)

Fluoxetine, Citalopram

Antidepressant

HPLC/MS

(42)

Paroxetine, Venlafaxine Paroxetine

Antidepressant

HPLC and GC/MS

(41)

and 12 other

Sisomicin, Astromicin

Antibiotic

HPLC

(47)

Netilmicin, Micronomicin

Antibiotic

HPLC/MS

(51)

Metronidazole, Spiramycin

Amphotericin B

Antibiotic

HPLC

(52)

5-Fluorouracil

Antineoplastic

HPLC

(56)

Irinotecan

Antineoplastic

HPLC

(57)

Docetaxel, Paclitaxel

Antineoplastic

HPLC

(58)

Imatinib

Antineoplastic

HPLC

(59)

Doxorubicin and other

Antineoplastic

HPLC

(60)

Ifosfamide

Antineoplastic

GC/MS

(62)

MS, mass spectrometry.

MS, mass spectrometry.

phenytoin in patient's sera utilized N, N-dimethyl derivatives of these drugs and "on-column" derivatization technique (17). Atwell et al. developed an HPLC assay for determination of phenobarbital and phenytoin in plasma using a porous particle silicic acid column. The mobile phase was composed of chloroform dioxane-isopropanol-acetic acid (310:9:7:1.0:0.1 by volume). The elution of drugs was monitored at 254 nm (18). Later, commercially available immunoassays took the place of these techniques for routine monitoring of anticonvulsants in clinical laboratories. Good correlations were observed between results obtained by immunoassays and GC analysis of phenytoin, phenobarbital, primidone, carbamazepine, and ethosuximide. Moreover, precision observed in these immunoassays for anticonvulsants was superior to GC analysis, and immunoassays were also relatively free from interfering substances (19). However, immunoassays also have certain limitations. Carbamazepine is metabolized to an active metabolite carbamazepine 10, 11-epoxide. This active metabolite may accumulate in patients with renal impairment and therapeutic monitoring of carbamazepine 10, 11-epoxide along with carbamazepine is recommended in certain patient populations. Currently, there is no commercially available immunoassay in the market to measure concentration of this metabolite. Moreover, cross-reactivity of this metabolite with carbamazepine immunoassays varied from very low (0-4%) to very high (94%) as discussed in Chapter 7. Berg and Buckley described an HPLC protocol for simultaneous determination of carbamazepine, carbamazepine 10, 11-epoxide, phenytoin, and phenobarbital in serum or plasma using a manual column-switching technique, an isocratic mobile phase, and UV detection. Diluted plasma or serum was injected directly to the system and reporting of results was achieved within 5min (20). A GC/MS method for simultaneous detection of carbamazepine and carbamazepine 10, 11-epoxide has also been reported. After microcolumn extraction of carbamazepine and its metabolite, the compounds were analyzed using a GC/MS. The capillary GC column used for the analysis was 25 m long with a 0.2-mm internal diameter and a film thickness of 0.33 ^m (cross-linked with 5% phenyl-methylsilicone) (21). A sensitive method for simultaneous determination of carbamazepine and its active metabolite carbamazepine 10, 11-epoxide has also been described using HPLC combined with tandem mass spectrometry. After liquid-liquid extraction, the specimen was analyzed using a Phenomenex Luna C18 column (150 x 2 mm, particle size 5 ^m) using a mobile phase composition of acetonitrile, methanol, and 0.1 % formic acid (10:70:20 by volume). Detection was achieved by a Micromass Quattro Ultima mass spectrometer (LC-MS-MS) using electrospray ionization, monitoring the transition of protonated molecular ion for carbamazepine at m/z 237.05, and carbamazepine 10, 11-epoxide at m/z 253.09 to the predominant ions of m/z 194.09 and 180.04, respectively. Using only 0.5 mL plasma, authors achieved a detection limit of 0.722 ng/mL for carbamazepine and 5.15 ng/mL for carbamazepine 10, 11-epoxide (22).

For newer antiepileptic drugs, HPLC or HPLC combined with mass spectrometry is used because of lack of commercially available immunoassays. Bahrami and Mohammadi (23) described an HPLC protocol for analysis of gabapentin in human serum after derivatization with 4-chloro-7-nitrobenzofuran, fluorescent-labeling agent. In this process, sensitivity was improved compared with o-phthalaldehyde derivatization. Contin et al. (24) described an HPLC method for simultaneous determination of lamotrigine, oxcarbazepine monohydroxy derivative, and felbamate in human plasma using only 0.5 mL specimen and a reverse phase HPLC column with UV detection. Although most reports for determination of lamotrigine concentration use HPLC, lamot-rigine in serum can also be analyzed using GC/MS after extraction and conversion into teri-butyldimethylsilyl derivative (25). Berry and Millington (26) reported an HPLC method for determination of a new antiepileptic drug pregabalin using a C8 column and derivatization of pregabalin with picryl sulfonic acid.

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  • gilly
    How to hook a ms to a gc and hplc?
    1 year ago

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