Bis(trifluoromethyl)aryl derivatives for drug analysis by gas chromatography electron capture negative ion chemical ionization mass spectrometry. Application to the measurement of low levels of clonidine in plasma

A gas chromatographic mass spectrometric assay for clonidine in plasma with a detection limit of a few picograms per ml was required. The p-trifluoromethylbenzyl, pentafluorobenzyl and pentafluorobenzoyl derivatives of clonidine were synthesized and the electron capture negative ion chemical ionization mass spectra of these compounds show extensive fragmentation with prominent ions at m/z 35 and 37 due to the two chlorine atoms in the clonidine molecule. Selected ion monitoring of specific high mass ions in these mass spectra indicated that the required sensitivity could not be obtained with these derivatives. Several bis(trifluoromethyl)pyrimidines were synthesized and these compounds were found to give an intense negative ion current under conditions of resonance electron capture. Consequently, a derivative of clonidine containing a bis(trifluoromethyl)aryl group was synthesized by reacting the drug with 3,5-bis(trifluoromethyl)benzoyl chloride. The negative ion mass spectrum of the reaction product has a base peak at m/z 673 and, when this ion is specifically monitored, an amount of derivative equivalent to 1 picogram of clonidine can be detected. This allowed the development of an assay for clonidine in plasma with a precision of 8% (SD) at 50 pg ml-1, 22% (SD) at 20 pg ml-1 and a lower limit for quantitative determination of 10 pg ml-1. Plasma concentrations of clonidine in 10 subjects given a single 50 micrograms oral dose are reported.     

Gas chromatographic negative ion mass spectrometric assay of 2-dicyclopropylmethylamino-2-oxazoline (S-3341), a new antihypertensive drug

The quantification in plasma and urine of 2-dicyclopropylmethylamino-2-oxazoline (S-3341), a new antihypertensive drug is described using a sensitive gas chromatographic negative ion mass spectrometric method with ammonia as moderating gas. After a two-step extraction, derivatization is carried out with 3,5-bis(trifluoromethyl)benzoyl chloride and the abundance of the molecular ion (m/z 420) obtained is compared with that of the tetradeuterated standard (m/z 424). The low background due to the high mass and negative ion detection provides a detection limit of about 1 pg per injection. Oral administration of 1 or 2 mg S-3341 to patients gives a maximum concentration of 3.3 +/- 0.7 ng ml-1 and 7.6 +/- 2.0 ng ml-1 at 1.8 +/- 0.6 h and 1.4 +/- 0.7 h and an average elimination half-life of 6.7 h.     

Quantitative analysis of albuterol in human plasma by combined gas chromatography chemical ionization mass spectrometry

A highly sensitive and specific quantitative assay for the determination of albuterol in human plasma, based on selected ion monitoring gas chromatography chemical ionization mass spectrometry, has been developed. The [MH]+ ions from the tri-TMS derivatives of albuterol (m/z 456) and the internal standard (2H3)albuterol (m/z 459), were assayed simultaneously by selected ion monitoring. The lower limit of quantitation is 0.25 ng ml-1 and the average assay precision (CV) for albuterol concentrations ranging from 0.25 ng ml-1 to 25 ng ml-1 is approximately 4%. This method is currently being employed for the routine quantitation of albuterol in plasma following the administration of doses therapeutically effective to man.     

Quantitative analysis of 5-fluorouracil and 5,6-dihydrofluorouracil in plasma by gas chromatography mass spectrometry

5-Fluorouracil and 5,6-dihydro-5-fluorouracil were analysed in the plasma of patients by combined gas chromatography mass spectrometry. 5-Bromouracil was the internal standard. After extraction from plasma with an isopropanol-diethyl ether mixture (20/80) the components were pentylated and the derivatives produced extracted into diethyl ether. Electron impact mass spectrometry was used for the simultaneous quantitative determinations of 5-fluorouracil and 5,6-dihydro-5-fluorouracil (detection limit 10 ng ml-1 5-fluorouracil, 80 ng ml-1 5,6-dihydro-5-fluorouracil). Chemical ionization was utilized to measure 5,6-dihydro-5-fluorouracil concentrations less than 80 ng ml-1 (sensitivity 10 ng ml-1). The biological applicability of these two techniques was demonstrated by analysing plasma samples from patients after administration of 5-fluorouracil or 5'-deoxyfluorouridine by intravenous injections and infusions.     

Gas chromatography chemical ionization mass spectrometric analysis of diminazene in plasma

A quantitative and sensitive method was developed for the determination of diminazene in plasma. The assay involves the reduction of diminazene to 4-aminobenzamidine and 4-hydrazinobenzamidine. The latter is further reduced to give an additional mole of 4-aminobenzamidine which is extracted, acetylated and condensed with hexafluoroacetylacetone to form a volatile derivative that is subsequently analyzed by gas chromatography chemical ionization mass spectrometry. 4-Aminobenzylamidine was synthesized and used as an internal standard. The method is reproducible and its sensitivity limit using 1 ml of plasma is 0.1 microgram diminazene ml-1. This sensitivity limit is sufficient to detect plasma levels in cattle following therapeutic doses of the drug.     

Measurement of 5-fluoro-2'-deoxyuridine serum levels by gas chromatography chemical ionization mass spectrometry

Serum levels of 5-fluoro-2'-deoxyuridine in cancer treated patients were measured by gas chromatography mass spectrometry under chemical ionization conditions; 1-(2-deoxy-beta-D-lyxofuranosyl)-5-fluorouracil (3'-epi-5-fluoro-2'-deoxyuridine) was used as an internal standard. The drug and internal standard were quantitatively isolated from the serum sample by a mini-column anion exchange method and the extract permethylated using potassium-tert-butoxide in dimethylsulphoxide and methyl iodide. The derivatized nucleosides were then re-extracted from the reaction mixture and analysed on a glass capillary column coated with Superox-4. The column was coupled directly to the chemical ionization source of the mass spectrometer; NH3 was used as the reagent gas. The gas chromatographic effluent was monitored at m/z 289, the [MH]+ ion of the N,O-permethyl derivatives of 5-fluoro-2'-deoxyuridine and the internal standard. Recovery of 5-fluoro-2'-deoxyuridine from serum in the 0-1 microgram ml-1 concentration range averaged 93 +/- 2% (SD); a linear detector response was observed up to 50 ng 5-fluoro-2'-deoxyuridine ml-1. At the 10 ng ml-1 level, a within-run assay precision of 10% (CV) (n = 5) was found, while a detection limit of about 1 ng 5-fluoro-2'-deoxyuridine ml-1 of serum was attained. The method was applied to the measurement of disappearance curves of 5-fluoro-2'-deoxyuridine in the serum of treated patients.     

Simultaneous determination of 6-oxo-prostaglandin F1 alpha and 2,3-dinor-6-oxo-prostaglandin F1 alpha in biological fluids by stable isotope dilution and negative ion chemical ionization mass spectrometry

A stable isotope dilution assay for the simultaneous determination of two metabolites of prostacyclin (1), 6-oxo-prostaglandin F1 alpha (2a) and 2,3-dinor-6-oxo-prostaglandin F1 alpha (3a), in human seminal fluid and human urine is described. A new chemical total synthesis of deuterated internal standard, 18,18,19,19-(2H4)-2,3-dinor-6-oxo-PGF1 alpha (3b), is presented and enables specific and sensitive quantification based on negative ion chemical ionization mass spectrometry. 2a and 3a were analysed as their methoxime pentafluorobenzyl ester tris(trimethylsilyl) ether derivatives in the selected ion monitoring mode registrating the [M-181]- fragments with a detection limit for both prostanoids of 10 pg per injection. The two metabolites occur in human seminal fluid in very low concentrations (2a: 2.8 ng ml-1; 3a: 1.7 ng ml-1) and cannot contribute significantly to the urinary metabolite levels which are in the range of 108-265 ng/24 h for 3a and 124-574 ng/24 h for 2a.     

Quantitative measurement of delta 9-tetrahydrocannabinol and two major metabolites in physiological specimens using capillary column gas chromatography negative ion chemical ionization mass spectrometry

delta 9-Tetrahydrocannabinol and two of its metabolites, 11-hydroxy-delta 9-tetrahydrocannabinol and 11-nor-9-carboxy-delta 9-tetrahydrocannabinol, can be measured in a single 1-ml sample of blood, plasma, or urine by a new assay which combines a relatively rapid extraction procedure with capillary column gas chromatography and negative ion chemical ionization mass spectrometry. Deuterium-labeled analogs of each cannabinoid are added to the physiological specimen as internal standards. Two extracts are obtained from each sample: a neutral fraction containing delta 9-tetrahydrocannabinol and 11-hydroxy-delta 9-tetrahydrocannabinol, and an acid fraction containing 11-nor-9-carboxy-delta 9-tetrahydrocannabinol. The neutral fraction is derivatized by treatment with trifluoroacetic anhydride; the acid fraction is first treated with BF3-methanol followed by reaction with trifluoroacetic anhydride. Under electron-capture chemical ionization conditions the derivatized delta 9-tetrahydrocannabinol and 11-nor-9-carboxy-delta 9-tetrahydrocannabinol give abundant molecular anions ideally suited for selected ion monitoring. The negative ion chemical ionization spectrum of the HO-THC-trifluoroacetate shows no molecular anion. Consequently, quantitation of the hydroxy metabolite is achieved by monitoring a fragment ion formed by loss of CF3CO2 from its molecular anion. The limits of reliable measurement are judged to be 0.1 ng ml-1 for 11-nor-9-carboxy-delta 9-tetrahydrocannabinol, 0.2 ng ml-1 for delta 9-tetrahydrocannabinol and 0.5 ng ml-1 for 11-hydroxy-delta 9-tetrahydrocannabinol. Four examples are given of the application of the assay to the analysis of specimens of medico-legal importance.     

Determination of CGS 16617 and stable isotope-labeled CGS 16617, an angiotensin-converting enzyme inhibitor, in human plasma by gas chromatography/mass spectrometry

An analytical method has been developed for the simultaneous determination of a novel orally active angiotensin-converting enzyme inhibitor (CGS 16617) and a stable isotope-labeled analog. Both compounds are isolated from human plasma using an ion-exchange column, derivatized with pentafluoropropionic anhydride and pentafluoropropanol, and analyzed by gas chromatography/mass spectrometry. After splitless injection on a methyl-silicon column, the compound is detected using negative ion chemical ionization with nitrous oxide as a reagent gas. CGS 16617 labeled with four deuteriums and two 13C is used as an internal standard. The accuracy and precision of the method, expressed as the overall mean +/- SD recovery obtained from two sets of 36 quality-control samples used during a clinical study (concentration range 0.2-100 ng ml-1 plasma), was 96.1 +/- 16.2% for unlabeled drug and 97.6 +/- 14.4% for the D4-labeled drug (concentration range 0.2-100 ng ml-1 plasma). The limit of quantification using 1 ml plasma is 0.2 ng ml-1 for both labeled and unlabeled drug.     

Quantitative determination of nitroglycerin in human plasma by capillary gas chromatography negative ion chemical ionization mass spectrometry

A quantitative method for determination of nitroglycerin in human plasma was developed. Nitroglycerin and the internal standard (butane-1,2,4-triyl trinitrate) were extracted from plasma with pentane. The extracts were analysed by gas chromatography mass spectrometry using fused silica capillary columns and electron capture negative ion chemical ionization. The quantitation limit of the method was about 50 pg ml-1. Linear calibration curves were obtained in the range of 50-1600 pg ml-1. Precision at the level of 100 pg ml-1 was 4%.     

Stereospecific gas chromatographic/mass spectrometric assay of the chiral labetalol metabolite 3-amino-1-phenylbutane.

We previously identified 3-amino-1-phenylbutane (APB) as an oxidative N-dealkylated, metabolite of the antihypertensive agent labetalol. Labetalol has two asymmetric centers and is used clinically as a mixture of the four possible stereoisomers; APB has one asymmetric center. We now report an enantiospecific gas chromatographic/mass spectrometric assay for APB in urine. After adding the internal standard 1-methyl-2-phenoxyethylamine and alkalinizing, the urine samples were extracted with ether. The extracts were derivatized with the optically active acid chloride prepared from (S)-alpha-methoxy-alpha-trifluoromethylphenylacetic acid. The derivatives were separated by capillary gas chromatography and detected by electron capture negative ion chemical ionization mass spectrometry with selected ion monitoring. The derivative of the R enantiomer eluted first, and the [M--32]- ions were monitored for both the drug and the internal standard. The method was linear in the 0.05-2.5 micrograms enantiomer-1 ml-1 range and had inter-assay and intra-assay coefficients of variation of less than 6%. The assay was used in the analysis of urine samples from a patient in labetalol therapy and no interference was found. Further studies are needed to elucidate the oxidative metabolism of labetalol and its stereochemical aspects.     

Selective and rapid liquid chromatography-mass spectrometry method for the determination of lercanidipine in human plasma

A specific liquid chromatography-mass spectrometric (LC-MS/MS) assay was developed and validated for the determination of lercanidipine, a dihydropyridine calcium channel blocker, in human plasma. Lercanidipine R-D3 was used as internal standard (IS). The drug was extracted from plasma using liquid-liquid extraction technique utilizing hexane: ethyl acetate as extraction solvent. The samples were analyzed using a prepacked Thermo Hypersil C(8) column and a mobile phase composed of a mixture of aqueous acetic acid and triethylamine in methanol. An ion trap mass spectrometer equipped with electrospray ionization (ESI) source operating in the positive ion mode was used to develop and validate the method. The method was proved to be sensitive and specific by testing six different human plasma batches. Linearity was established for the concentration ranges of 0.1-16 ng/ml with a regression factor of 0.9996. The lower limit of quantitation was identifiable and reproducible at 0.1 ng/ml with a precision of 7.2%.     

Enantioselective LC/MS method for the determination of an antimalarial agent Fenozan B07 in dog plasma

A chiral liquid chromatography/mass spectrometry (LC/MS) bioanalytical procedure has been developed for the analysis of the antimalaric agent Fenozan B07 in dog plasma. Normal-phase chromatography involving a phenylcarbamate derivative of cellulose coated on silica gel as the chiral stationary phase was used to resolve (-)-(S,S)-B07 from (+)-(R,R)-B07. The enantiomers were detected by a mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) interface operated in the negative ion mode. A mass spectrum, characterized by a base peak of m/z 285, was obtained for each enantiomer. The m/z 285 ion was very specific for the analysis of both enantiomers in the plasma. The selected ion monitoring analysis of the plasma samples was therefore performed at m/z 285 for quantitative purposes. The enantiomers were extracted from the plasma in a basic medium and purified by solid-phase extraction using a hydrophilic-lipophilic balanced sorbent. A lower limit of quantification of 2 ng/mL in plasma was achieved for both enantiomers. The quantitative procedure reported in this study was highly specific and sensitive, and was validated according to the FDA guidance on bioanalytical method validation.     

The determination of terbutaline in human plasma by selected ion monitoring of the t-butyldimethylsilyl ether

A method is described for the quantitative determination of terbutaline in 2 ml human plasma. The drug is extracted from plasma as the terbutaline tetraphenylboron ion pair and determined by gas chromatography mass spectrometry of its t-butyldimethylsily ether. Salbutamol is used as internal standard. Quantification is achieved by selected ion monitoring of the ion m/z 482 derived from t-butyldimethylsilyl terbutaline and m/z 495 from t-butyldimethylsilyl salbutamol. The detection limit was estimated to be 250 pg terbutaline ml-1 plasma. The coefficient of variation at the level of 1 ng terbutaline ml-1 was 4.1% (n = 5).     

Method for the simultaneous determination of losartan and its major metabolite, EXP-3174, in human plasma by liquid chromatography–electrospray ionization tandem mass spectrometry

A liquid chromatography–electrospray ionization tandem mass spectrometric method was developed for the simultaneous determination of losartan and its major active metabolite, EXP-3174, in human plasma. The two analytes and the internal standard (DuP-167) were extracted from plasma under acidic conditions by using solid-phase extraction cartridges containing a sorbent of copolymer, poly(divinylbenzene-co-N-vinylpyrrolidone). The analytes were separated by LC equipped with a reversed-phase C₁₈ column, and introduced into the mass spectrometer via the electrospray ion source with pneumatically-assisted nebulization. For LC–MS–MS samples, an isocratic mobile phase consisting of [0.1% triethylamine–0.1% acetic acid (pH 7.1)]–acetonitorile (65:35, v/v) was used, and the assay was monitored for the negative fragment ions of the analytes. The method demonstrated linearity from 1 to 1000 ng/ml for both losartan and EXP-3174. The limit of quantification for both compounds in plasma was 1 ng/ml. This assay method may be useful for the measurement of levels of the two compounds in clinical studies of losartan.     

Quantification of retinoic acid by gas-liquid chromatography-mass spectrometry: total versus all-trans-retinoic acid in human plasma

An assay based on negative ion chemical ionization mass spectrometry has been developed to quantify retinoic acid in plasma or serum. The lower limit of detection is 75 pg (240 fmol); normal values of retinoic acid can be determined on as little as 40 microliters of human plasma. The plasma concentrations of total retinoic acid in 12 healthy male volunteers taking no medication or vitamin supplementation ranged from 2.8 to 6.6 ng/ml; the mean was 4.9 ng/ml. The assay can be manipulated to measure all-trans-retinoic acid alone; about 75% of retinoic acid in human plasma or rat serum is all-trans-retinoic acid. Both retinol and retinoic acid can be quantified on the same 0.1-ml sample; the concentration of retinoic acid in human plasma or rat serum is at least 150-fold less than that of retinol.     

Determination of phenelzine in human plasma with gas chromatography—mass spectrometry using an isotope labeled internal standard

A quantitative gas chromatographic—mass spectrometric assay was developed for the determination of phenelzine in human plasma. Phenelzine, in aqueous solution or in plasma reacts at room temperature with pentafluorobenzaldehyde to form quantitatively a hydrazone derivative. The derivative has good gas chromatographic characteristics. The assay utilizes selected ion monitoring in a gas chromatographic effluent, the molecular ion generated by electron impact ionization of phenelzine derivative. Phenelzine-d₇ was synthesized and used as an internal standard. The assay can measure 2 ng/ml of the drug with about 10% precision.The method was used for the determination of steady state levels of phenelzine in the plasma of patients taking a therapeutic dose of the drug.     

A sensitive assay for the quantitative analysis of vinorelbine in mouse and human EDTA plasma by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry

A sensitive, specific and fast high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) assay for the determination of vinorelbine in mouse and human plasma is presented. A 200 microL aliquot was extracted with solid-phase extraction (SPE) using Bond-Elut C(2) cartridges. Dried extracts were reconstituted in 100 microL 1 mM ammonium acetate pH 10.5-acetonitrile-methanol (21:9:70, v/v/v) containing the internal standard vintriptol (100 ng/mL) and 10 microL volumes were injected onto the HPLC system. Separation was achieved on a 50 mm x 2.0 mm i.d. Gemini C(18) column using isocratic elution with 1 mM ammonium acetate pH 10.5-acetonitrile-methanol (21:9:70, v/v/v) at a flow rate of 0.4 mL/min. HPLC run time was only 5 min. Detection was performed using positive ion electrospray ionization followed by tandem mass spectrometry (ESI-MS/MS). The assay quantifies vinorelbine from 0.1 to 100 ng/mL using human plasma sample volumes of 200 microL. With this method vinorelbine can be measured in mouse plasma samples when these samples are diluted eight times in control human plasma. Calibration samples prepared in control human plasma can be used for the quantification of the drug. The lower limit of quantification in mouse plasma is 0.8 ng/mL. This assay is used to support preclinical and clinical pharmacologic studies with vinorelbine.     

Rapid and sensitive LC–MS/MS assay for the quantitation of 20(S)-protopanaxadiol in human plasma

This paper describes a rapid and sensitive method for the quantitation of 20(S)-protopanaxadiol (PPD) in human plasma based on high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS). The analyte and internal standard (I.S.), ginsenoside Rh₂, were extracted from plasma by liquid–liquid extraction and separated on a Zorbax extend C₁₈ analytical column using methanol–acetonitrile-10 mM ammonium acetate (47.5:47.5:5, v/v/v) as mobile phase. Detection was by tandem mass spectrometry using electrospray ionization in the positive ion mode and multiple reaction monitoring (MRM). The assay was linear over the concentration range 0.1–100.0 ng/ml with a limit of detection of 0.05 ng/ml. The method was successfully applied to a clinical pharmacokinetic study in healthy volunteers after a single oral administration of a PPD 25 mg capsule.     

Determination of palmatine in canine plasma by liquid chromatography-tandem mass spectrometry with solid-phase extraction

A sensitive and specific high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) method has been developed and validated for the determination of palmatine in canine plasma. Palmatine and jatrorrhizine (internal standard, I.S.) were extracted from plasma samples by solid-phase extraction (SPE) using Oasis HLB cartridges. The chromatographic separation was performed on a Waters XTerra MS C(18) reversed-phase column at 30 degrees C. The gradient mobile phase, delivered at 0.25 mL/min, was composed of a mixture of acetonitrile -0.1% (v/v) acetic acid aqueous solution adjusted to pH 2.8 with triethylamine. Positive electrospray ionization was utilized as the ionization source. Palmatine and the internal standard (I.S.) were determined using multiple reaction monitoring (MRM) of precursor-->product ion transitions at m/z 352-->336 and m/z 338-->322, respectively. The lower limit of quantification (LLOQ) was 0.1 ng/mL using 100 microL plasma samples and the linear calibration range was from 0.1 to 500 ng/mL. The inter-day and intra-day RSDs were lower than 9.9% and the recoveries of palmatine ranged from 87.3 to 100.9%. The mean extraction recoveries of palmatine and the I.S. were 99.2 and 96.8%, respectively. The method has been successfully applied to the pharmacokinetic studies of palmatine in beagle dogs after oral administration and intramuscular injection of palmatine.