Design of a stable isotope dilution gas chromatography/mass spectrometric assay for cAMP: Comparison with standard protein-binding and radioimmunoassay methods

A stable isotope dilution assay is presented in which picomole quantities of cAMP can be determined with high precision and selectivity using gas chromatography and electron impact mass spectrometry with multiple ion detection techniques. Using synthetic [2,8-²H₂,6-¹⁵N]-cAMP as the internal standard, suitable specificity was obtained by monitoring the (MCH₃)⁺ fragment ions of the trimethylsilyl derivatives of cAMP and the internal standard at $\text{m}\text{z}$ 530 and $\text{m}\text{z}$ 533, respectively. The sensitivity of the assay as judged from the lower limit of detection of the mass spectrometer was 3.0 pmol. Rat liver and human urine cAMP levels were assayed using gas chromatography/mass spectrometry and were compared with levels determined by protein-binding assays and radioimmunoassays for the same samples. The intraassay coefficients of variation of the gas chromatography/mass spectrometry assay were 5.3% for the rat liver sample (cAMP level 832 pmol/g) and 6.0% for the urine sample (cAMP level 2.50 μmol/liter). Comparison of the levels of cAMP determined by the three assay methods showed correlation to within 10% variation.     

Analysis of histamine and N tau-methylhistamine in plasma by gas chromatography-negative ion-chemical ionization mass spectrometry

Current methods of quantitation of histamine and its major metabolite N tau-methylhistamine are inaccurate and insensitive to the very low concentrations that exist in plasma samples. Therefore, an accurate and sensitive method for quantification in plasma has been developed using the stable isotope dilution assay with negative ion-chemical ionization mass spectrometry. For histamine, after the addition of [2H4]histamine to 2 ml of plasma, the plasma sample is deproteinized, extracted into butanol, back extracted into HCl, derivatized to the pentafluorobenzyl derivative (CH2C6F5)3-histamine, purified on silica gel columns, and then quantified with negative ion-chemical ionization mass spectrometry by selected ion monitoring of the ratio of ions m/z 430/434. For N tau-methylhistamine, after the addition of N tau-[2H3]methylhistamine to 2 ml of plasma, the plasma sample is deproteinized, extracted into butanol, back extracted into HCl, derivatized to the heptafluorobutyryl derivative (C3F7CO2)2-N tau-methylhistamine, purified on silica gel columns, and then quantified with negative ion-chemical ionization mass spectrometry by selected ion monitoring of the ratio of ions m/z 497/500. The precision of the histamine assay is 3.1% and the accuracy is 95.5 +/- 2.5% while the precision of the N tau-methylhistamine assay is 1.9% and the accuracy is 106.8 +/- 1.9%. The lower limits of sensitivity are 1 pg for histamine and 6 pg for N tau-methylhistamine injected on column. Using the assay in three normal human volunteers, plasma concentrations of histamine were 130, 92, and 85 pg/ml, and of N tau-methylhistamine were 229, 228, and 216 pg/ml. This assay provides a very sensitive and accurate method of quantitation of histamine and N tau-methylhistamine in plasma samples.     

Mass spectrometric analysis of cytokinins in plant tissue VII. Quantification of cytokinin bases by negative ion mass spectrometry

A study of derivatives of N⁶-(isopent-2-enyl)adenine formed by substitution at N-9 indicated that sensitivity of detection by chemical ionization mass spectrometry was maximized by a pentafluorobenzyl substituent and negative ion monitoring. O-t-Butyldimethylsilyl-9-pentafluorobenzyl derivatives of zeatin (Z),cis-zeatin (cis-Z), and dihydrozeatin (DZ) were characterized by mass spectrometry. A procedure was based on these stable derivatives and negative ion chemical ionization mass spectrometry for quantification of zeatin and dihydrozeatin in plant tissue.     

I. A unique deaminated metabolite of sulfamethazine [4-amino-N-(4, 6-dimethyl-2-pyrimidinyl) benezenesulfonamide] in swine

¹⁴C-Sulfamethazine [4-amino-$\text{N}$-(4, 6-dimethyl-2-pyrimidinyl)- ¹⁴C-benzenesulfonamide] was administered orally to young, 21-kg male swine. A unique metabolite, $\text{N}$-(4, 6-dimethyl-2-pyrimidinyl)- ¹⁴C-benzenesulfonamide (¹⁴C-desaminosulfamethazine), was identified in skeletal muscle collected 24 hr after dosing. The structure of the metabolite was confirmed by synthesis, comparative electron impact mass spectrometry, chemical ionization mass spectrometry, and infrared spectroscopy.     

Mass spectrometric analysis of cytokinins in plant tissue VII. Quantification of cytokinin bases by negative ion mass spectrometry

A study of derivatives of N⁶-(isopent-2-enyl)adenine formed by substitution at N-9 indicated that sensitivity of detection by chemical ionization mass spectrometry was maximized by a pentafluorobenzyl substituent and negative ion monitoring. O-t-Butyldimethylsilyl-9-pentafluorobenzyl derivatives of zeatin (Z),cis-zeatin (cis-Z), and dihydrozeatin (DZ) were characterized by mass spectrometry. A procedure was based on these stable derivatives and negative ion chemical ionization mass spectrometry for quantification of zeatin and dihydrozeatin in plant tissue.For part VI, see Letham and Singh (1989).     

Isolation and derivatization of plasma taurine for stable isotope analysis by gas chromatography-mass spectrometry

A method for the isolation and derivatization of plasma taurine is described that allows stable isotope determinations of taurine to be made by gas chromatography-mass spectrometry (gc-ms). The isolation procedure can be applied to 0.1 ml of plasma: the recovery of plasma taurine was 70–80%. For gc separation, taurine was converted to its dimethylaminomethylene methyl ester derivative which could not be detected by hydrogen flame ionization, but could be monitored readily by NH₃ chemical ionization mass spectrometry. The derivatization reaction occurred partially on-column and required optimization of injection conditions. Using stable isotope ratiometry multiple ion detection, $\text{[M + 2 + H]}{}^{\mathrm{+}}\text{[M + H]}{}^{\mathrm{+}}$ ion ratio of natural abundance taurine was determined with a standard deviation of less than ±0.07% of the ratio. The [1,2-¹³C]taurine/taurine mole ratios of standard mixtures could be accurately determined to 0.001. This stable isotope gc-ms method is suitable for studying the plasma kinetics of [1,2-¹³C]taurine in infants who are at risk with respect to taurine depletion.     

Determination of 6-keto-PGF1α, 2,3-dinor-6-keto-PGF1α, thromboxane B2, 2,3-dinor-thromboxane B2, PGE2, PGD2 and PGF2α in human urine by gas chromatography—negative ion chemical ionization mass spectrometry

A method for quantification of 6-keto-PGF_1α, 2,3-dinor-6-keto-PGF_1α, TXB₂, 2,3-dinor TXB₂, PGE₂, PGD₂ and PGF_2α in human urine samples, using gas chromatography—negative ion chemical ionization mass spectrometry, is described. Deuterated analogues were used as internal standards. Methoximation was carried out in urine samples which were subsequently applied to phenylboronic acid cartridges, reversed-phase cartridges and thin-layer chromatography. The eluents were further derivatized to pentafluorobenzyl ester trimethylsilyl ethers for final quantification by gas chromatography—mass spectrometry. The overall recovery was 77% for tritiated 6-keto-PGF_1α and 55% for tritiated TXB₂. Urinary levels of prostanoids were determined in a group of six volunteers before and after intake of the thromboxane synthase inhibitor Ridogrel, and related to creatinine clearance.     

Femtomole level of analysis of biogenic amines and amino acids using functional group mass spectrometry

A general method for the determination of compounds possessing either the primary amine structure, R-CH₂-NH₂ (I), or the α-amino acid structure, RCHNH₂COOH (II), has been devised using gas chromatography and mass spectrometry. Trimethylsilyl derivatives of the biogenic amines (phenylethylamines, indoleethylamines, or Ω-amino acids) produce an intense ion at $\text{m}\text{e}$ 174 upon fragmentation; TMS derivatives of α-amino acids produce an ion at $\text{m}\text{e}$ 218. For maximum sensitivity, chromatograms were obtained with the mass spectrometer tuned to detect a single ion fragment characteristic of a group of structurally related compounds (i.e., functional group GC-MS). At $\text{m}\text{e}$ 174 up to 14 compounds of Type I, including glycine, γ-aminobutyric acid, dopamine, and 5-hydroxytryptamine, could be determined in a single analysis. Detection limits range from 10–100 femtomoles (10^?15 moles). At $\text{m}\text{e}$ 218, eight compounds of Type II, including isoleucine, phenylalanine, tyrosine, and DOPA could be determined. This technique has been applied to the assay of these compounds in extracts containing 0.1 mg mouse brain or abdominal ganglia of the marine molluse, Aplysia californica.     

Analysis of microquantities of choline and its esters utilizing gas chromatography-chemical ionization mass spectrometry.

Gas chromatography-chemical ionization mass spectrometry has been applied successfully in the analysis of choline and its esters. This approach serves to extend further the potential of existing gas chromatographic procedures which are capable of the microestimation of choline esters following their N-demethylation by either chemical or physical means. Typical fragmentation patterns with ions at $\text{m}\text{e}\text{= 72}$ and $\text{m}\text{e}\text{= (M + 1)}$ were obtained for each choline ester derivative. When methane was used as the reactant gas, the above fragments were approximately of equal abundance for each ester. Use of isobutane as reactant gas yielded almost 80% of the (M + 1) fragment, and only approximately 5% of the fragment ion at $\text{m}\text{e}\text{= 72}$. Recovery of all fragments was linear for nondeuterated as well as deuterated analogs of choline ester derivatives. Recovery, as evident from the analysis of records of relative ratios of injected isotopic variants of these esters, indicated that this analysis of choline esters using chemical ionization mass spectrometry coupled with gas chromatography is quantitative and highly reproducible.     

Metabolism of prostacyclin in cynomolgus monkey

The metabolism of prostacyclin (PGI₂) $\text{in vivo}$ was investigated in Cynomolgus monkey. Following intravenous infusion of 11-[³H]-PGI₂ for three days, pooled urine was extracted with Amberlite XAD-2, then chromatographed and purified by Sephadex LH-20, and reverse phase column chromatography. Radioactive fractions were converted to appropriate derivatives for identification by gas chromatography mass spectrometry. Twelve metabolites were characterized, the major of which was 6-keto-PGF_1α, accounting for 13% of the urinary radioactivity. The metabolic pathways are similar to those observed earlier in the rat. The excretion of substantial amounts of unchanged 6-keto-PGF_1α indicated that the monkey was not able to metabolize PGI₂ as avidly as the rat.     

Quantitative measurement of a new synthetic hetrazepine derivative, BN50730, in human plasma and urine by combined liquid chromatography—negative chemical ionization mass spectrometry using a particle beam interface

A new simple and sensitive assay has been developed for the quantitative measurement of BN50730 at the picomole level in human plasma and urine. The drug and the internal standard (BN50765) were measured by combined liquid chromatography—negative chemical ionization mass spectrometry with methane as the reagent gas. A simple solid—liquid extraction procedure was used to isolate BN50730 from complex biological matrices. Mild operating conditions were required to assay the parent drug with a particle beam interface from Hewlett-Packard. The mass spectrometer was tuned to monitor the intense ion m/z 333, which was generated in the ion source by a dissociative capture process. This assay was performed with 1 ml of plasma or 0.1 ml of urine, and the quantification limit of the method was statistically calculated as 1 ng ml⁻¹. The very low relative standard deviation and mean percentage of error calculated during the different within-day or between-day repeatability assays clearly demonstrate the ruggedness of the technique for the routine determination of BN50730 in the biological fluids. Some preliminary results on the pharmacokinetics of the drug are presented to illustrate the applicability of this new powerful LC—MS method.     

Tandem mass spectrometry for on-line fermentation monitoring

Summary Membrane introduction mass spectrometry has been employed for on-line determination of the major products and volatile metabolites ofBacillus polymyxa fermentation. Samples were introduced into the mass spectrometer via a direct insertion membrane probe in which the aqueous solution flowed past a membrane located in the ion source of the mass spectrometer. Concentrations of the products 2,3-butanediol, acetoin, ethanol and acetic acid in fermentation broth were measured by tandem mass spectrometry after permeation through the membrane and ionization by chemical ionization. External standards were employed for quantification and a large linear response range was available for each of the major products observed. Dissolved CO₂ and O₂, as well as CO₂ in the off gases, were also monitored on-line by mass spectrometry. The use of tandem mass spectrometry has allowed the identification of products that were not previously known to be present in measurable amounts.     

A new class of lipoxygenase inhibitor. Polyunsaturated fatty acids containing sulfur

A series of unsaturated and polyunsaturated fatty acids with a sulfur atom substituting for a methylene unit of the chain has been prepared and characterized. The syntheses were accomplished by the Wittig coupling of the ylid derived from the triphenylphosphonium salt of 9-bromononanoic acid with aldehydes containing sulfur. The newly formed double bond had predominately the natural Z geometry even when the starting aldehyde was conjugated with the sulfur atom. The sulfides 13-thia-9(Z)-octadecenoic acid ($\text{2}$), 13-thia-9(Z), 11(E)-octadecadienoic acid ($\text{5}$) and 13-thia-9(E), 11(E)-octadecadienoic acid ($\text{6}$) were readily converted into their sulfoxide derivatives by treatment with an equivalent amount of m-chloroperoxybenzoic acid. The structures of the novel compounds were confirmed by the application of ir, uv, ¹H-nmr, ¹³C-nmr, and (as methyl esters) chemical ionization mass spectrometry. Two members of this new family of fatty acids ($\text{5}$ and $\text{6}$) were found to inhibit the catalysis of the oxygenation of linoleic acid by soybean type-1 lipoxygenase. The analysis of the kinetic data for compound $\text{5}$ indicated that the type of inhibition was reversible competitive with an inhibition constant of 30 μM.     

Application of selected ion monitoring technique for quantitative determination of picomole levels of pyrrolidine in the brain

A specific and sensitive method for analysis of brain pyrrolidine, a volatile amine with potent synaptotropic actions on the peripheral and central nervous systems, was devised. The method involves the isolation of volatile amines by steam distillation and the qualification and quantification of pyrrolidine by gas chromatography/mass spectrometry ($\text{gc}\text{ms}$) including a selected ion monitoring technique with deuterium-labeled pyrrolidine as an internal standard. The lower limit of quantification for the method was 2 pmol, and the mean concentration of pyrrolidine in the rat whole brain was determined to be 95 pmol/g of tissue.     

Enzymatic formation of (20R, 22R)-20,22-dihydroxycholesterol from cholesterol and a mixture of 16O2 and 18O2: random incorporation of oxygen atoms

[4-¹⁴C]Cholesterol was incubated with an adrenocortical preparation in the presence of ¹⁶O₂ and ¹⁸O₂ devoid of significant ¹⁶O¹⁸O. Isolated (20R,22R)-20,22-dihydroxycholesterol was converted to a trimethylsilyl derivative and analyzed by gas chromatography - mass spectrometry to determine the isotope distribution of the oxygen atoms at C-20 and C-22. The ions of $\text{m}\text{e}$ 289, 291, and 293 (comprising the C₈ C-20 to C-27 side-chain and containing, respectively, ¹⁶O₂, ¹⁶O¹⁸O, and ¹⁸O₂) exhibited a binomial distribution indicating that the oxygen atoms of the vicinal glycol were drawn at random from the atomic pool of the oxygen molecules. If both side-chain hydroxyl groups had originated from the atoms of the same oxygen molecule, the ion of $\text{m}\text{e}$ 291 would have been absent.     

Determination of free salsolinol concentrations in human urine using gas chromatography—mass spectrometry

The urine concentrations of free salsolinol were determined in six healthy volunteers, using a gas chromatographic—mass spectrometric method with electron-capture negative-ion chemical ionization after derivatization with pentafluoropropionyl anhydride. The sensitivity of this method allows the quantification of salsolinol concentrations of 0.55 pmol/ml. The synthesis of [²H₄]salsolinol from dopamine and [²H₄]acetaldehyde via a Pictet—Spengler condensation is described; [²H₄]salsolinol was used as the internal standard for salsolinol quantification. The urine concentrations of free salsolinol ranged from ca. 1 to 6 pmol/ml.     

Assay of liver microsomal cholesterol 7 alpha-hydroxylase using deuterated carrier and gas chromatography-mass spectrometry

The activity of cholesterol 7α-hydroxylase in rat liver microsomes was assayed by measuring the mass of 5-cholestene-3β, 7α-diol formed from endogenous cholesterol under standardized incubation conditions. After termination of incubations, a known amount of 5-[24,25,7β-²H₃]cholestene-3β,7α-diol was added. A chloroform extract of the incubation mixture was subjected to thin layer chromatography and the fraction containing 5-cholestene-3β,7α-diol was converted into trimethylsilyl ether. The trimethylsilyl ether was subjected to combined gas chromatography-mass spectrometry and the amount of unlabeled 5-cholestene-3β,7α-diol in the mixture was calculated from the ratio between the relative intensitics of the peaks at $\text{m}\text{e}$ 456 (M-90) and $\text{m}\text{e}$ 459 [M-(90 + 3)]. The precision of the method was ±2.2% (SD). The results with this method of assay of cholesterol 7α-hydroxylase were compared with those obtained with a method based on conversion of a trace amount of added [4-¹⁴C]cholesterol into 5-cholestene-3β,7α-diol.     

Determination of iodide in urine using electrospray ionization tandem mass spectrometry

A rapid and sensitive electrospray ionization (ESI) tandem mass spectrometry (MS–MS) procedure was developed for the determination of iodide (I⁻). A gold (Au) and I⁻ complex was formed immediately after the addition of the chelating agent NaAuCl₄ to I⁻ solution, and was extracted with methyl isobutyl ketone. One to five microliters of the extract were injected directly into an ESI–MS–MS instrument. I⁻ quantification was performed by selecting reaction monitoring of the product ion I⁻ at m/z 127 derived from the precursor ion ¹⁹⁷AuI₂⁻ at m/z 451. I⁻ concentration was measured in the quantification range from 10⁻⁷ to 10⁻⁵ M using 50 μL of solution within 10 min. Iodate was reduced to I⁻ with ascorbic acid and determined. I⁻ concentration in reference urine 2670a was measured after treatments.     

Positive-negative, chemical-ionization, direct exposure mass spectrometry of underivatized prostaglandins

Nine underivatized prostaglandins were examined using direct exposure, ammonia, chemical-ionization, pulsed positive-negative ion mass spectrometry. The positive ion spectra were characterized by (M+18)⁺ ion adducts. The negative ion spectra were characterized by ions which dependence upon the functionality present in the cyclopentane ring system (acetal for TXB₂). The E and D series prostaglandins gave (M-18)⁻ as the major negative ion, while the F series and TXB₂ were characterized by negative ions corresponding to (M-1)⁻, and PGA₂ by the parent (M)⁻ ion. Prostaglandin 6-keto-PGF_1α was anomalous in this respect showing apparent dehydration, interpreted as an overall (M-18+1)⁺ and (M-18-1)⁻ in the positive and negative ion spectra, respectively. All major ion types were shown to give essentially a linear response with respect to concentration in the 10–1000 ng range. Although these initial studies were conducted under ideal conditions, it would appear that direct chemical ionization techniques show promise for providing direct structural information on prostaglandins without the need for prior chemical derivation.     

Metabolic activation of procarbazine

Procarbazine chemical degradation and rat $\text{in vitro}$ and $\text{in vivo}$ metabolism have been investigated. Procarbazine was rapidly oxidized to the azo derivative in aqueous solution in the presence of oxygen. $\text{In vitro}$ rat liver supernatany and microsomal preparations oxidize the azo function to azoxy isomers and further hydroxylate these metabolites in a manner that may by analogous to 1,2-dimethylhydrazine metabolism. The hydroxylated metabolites are activated species that chemically react to give methylating and alkylating agents. An additional metabolic pathway was observed $\text{in vivo}$. This suggests that procarbazine may be converted to free radical intermediates that decompose to give methane and N-isopropyltoluamide. Procarbazine metabolites have been separated and identified using high performance liquid chromatography and direct probe chemical ionization mass spectrometry.