Determination of nicotine and cotinine in rat plasma by liquid chromatography-tandem mass spectrometry

A method was developed for the efficient determination of nicotine and cotinine in rat plasma samples originating from nicotine exposure studies. Nicotine and cotinine were extracted from plasma samples with dichloromethane and concentrated to minimum volume with nitrogen stream. The volatility of nicotine was prevented by the addition of hydrochloric acid to the organic solvent during evaporation. The samples were analysed using liquid chromatography with triple quadrupole mass spectrometry. For quantification, the deuterated internal standards were added and the most intensive MS-MS ion of the analyte and internal standards were monitored. For confirmatory analysis, two specific MS-MS ions, viz. m/z 132 and 106 for nicotine and m/z 80 and 98 for cotinine, were monitored and the ratios between the ions were calculated and compared with those of standards. The ratios have to be within the tolerances of the EU criteria. The limit of identification of the developed method was 1 microg/l. The repeatability ranged from 5 to 12% (R.S.D.) for nicotine and from 3 to 5% for cotinine at the concentration level of 1-60 microg/l (n = 4).     

Electron-impact ionization detection of scopolamine by gas chromatography-mass spectrometry in rat plasma and brain

Scopolamine, a muscarinic receptor antagonist, was measured in rat plasma and brain using gas chromatography-mass spectrometry with electron-impact ionization detection. Extracted scopolamine was either directly derivatized or first hydrolyzed to scopine, then derivatized with heptafluorobutyric anhydride, separated on a capillary column and quantified by mass fragmentography. Electron-impact ionization produced a common fragment peak at m/z 138 that was monitored along with trideuterated scopolamine, an internal standard (m/z 141). The method can be used to measure scopolamine concentrations of 2 ng/ml in rat plasma and 20 ng/g in rat brain.     

A liquid chromatography-mass spectrometry method for nicotine and cotinine; utility in screening tobacco exposure in patients taking amiodarone

A liquid chromatographic mass spectrometric (LC-MS) assay for the quantification of nicotine and cotinine in human specimens was developed. Human serum and urine (100 μL) were subjected to liquid-liquid extraction. For glucuronidated cotinine, serum was alkalinized and hydrolyzed before extraction. The dried samples were reconstituted and run using gradient flow reverse-phase liquid chromatography with MS detection. The ions utilized for quantification of nicotine, cotinine and milrinone (internal standard) were 162.8, 176.9 and 211.9 m/z, respectively. The mean recoveries were over 80% for cotinine and nicotine with excellent linearity between nominal concentrations and peak area ratios, over a wide concentration range. The percentage coefficient of variation and mean error of the inter- and intra-day validations were <15% for nicotine and cotinine. Analysis of serum from cardiac patients receiving amiodarone suggested that a number of patients were either active smokers or exposed to second-hand smoke. Significant concentrations of nicotine and cotinine were measured in the urine of a known smoking volunteer. The method was highly specific, sensitive and applicable as a tool in detecting and monitoring the passive exposure to tobacco smoke using small specimen volumes (0.1 mL).     

Characterisation of nicotine and related compounds using electrospray ionisation with ion trap mass spectrometry and with quadrupole time-of-flight mass spectrometry and their detection by liquid chromatography/electrospray ionisation mass spectrometry

Electrospray ionisation ion trap mass spectrometry (ESI-MS(n)) has been used to study the fragmentation patterns of nicotine and nine of its related compounds. From this study certain characteristic fragmentations are apparent with generally the pyrrolidine or piperidine ring being subject to chemical modifications. The structures of the product ions proposed for the ESI-MS(n) study have been supported by results from electrospray ionisation quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS). Compounds with pyrrolidine and piperidine rings that possess an unsubstituted N atom have been shown to lose NH(3) at the MS(2) stage. Those compounds with N-methyl groups lose CH(3)NH(2) at the MS(2) stage. The loss of NH(3) or CH(3)NH(2) leaves the corresponding rings opened and this is followed by ring closure at the pyridine-2 carbon atom. Mono-N-oxides fragment in a similar way but the di-N-oxide can also fragment by cleavage of the bond between the pyridine and pyrrolidine rings. Cotinine also can undergo cleavage of this bond between the rings.This data therefore provides useful information on how substituents and the nature of the non-pyridine ring can affect the fragmentation patterns of nicotine and its related compounds. This information can be used in the characterisation of these compounds by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) which results in the separation of nicotine and its related compounds with limits of detection (LODs) ranging from 15 to 105 ng/mL. The use of LC/ESI-MS to study nicotine-containing samples resulted in the simultaneous and unambiguous identification of seven of the compounds discussed in this paper: cotinine identified at retention time 12.5 min (with its [M+H](+) ion at m/z 177), nornicotine 16.0 min (m/z 149), anatabine 18.0 min (m/z 161), myosmine 18.5 min (m/z 147), anabasine 20.4 min (m/z 163), nicotine 22.2 min (m/z 163), and nicotyrine 31.4 min (m/z 159). For quality control of nicotine replacement therapy products, these nicotine impurities can be readily identified and determined at levels up to 0.3% for single impurities and up to 1.0% for total impurities.     

Liquid chromatographic determination of honokiol and magnolol in hou po (Magnolia officinalis) as the raw herb and dried aqueous extract

A validated analytical method is described for the determination of honokiol and magnolol in Hou Po (Magnolia officinalis) as the dried raw herb and the commercially prepared dried aqueous extract. The samples were extracted with methanol by the Soxhlet method, and the extract was analyzed by liquid chromatography with photodiode array (LC/PDA) detection with confirmation of analyte identity by negative-ion electrospray ionization tandem mass spectrometry (ESI-MS/MS). A C18 column was used with a menthanol--0.1% aqueous acetic acid gradient mobile phase. Honokiol and magnolol were quantified at 288 nm. With the MS detector, the honokiol precursor ion at m/z 265 was shown to produce ions at m/z 222 and 224. For magnolol, the precursor ion at m/z 265 produced the ions at m/z 247 and 245. Comparable results were obtained for the LC/PDA and LC/ESI-MS/MS methods of quantitation. Six commercially prepared dried aqueous extracts were analyzed. The levels of honokiol and magnolol found in the raw herb were 17.0 and 21.3 mg/g, respectively. The limits of detection for honokiol and magnolol in the raw herb were 0.45 and 0.58 mg/g, respectively, and in the dried aqueous extract, 0.04 and 0.30 mg/g, respectively.     

Simultaneous determination of nicotine and its metabolite, cotinine, in rat blood and brain tissue using microdialysis coupled with liquid chromatography: pharmacokinetic application

To elucidate the disposition of nicotine in the brain is important because the neuropharmacological effects from nicotine exposure are centrally predominated. The aim of the present study was to develop a rapid and simple method for the simultaneous determination of unbound nicotine and its main metabolite, cotinine, in rat blood and brain tissue. We coupled a multiple sites microdialysis sampling technique with HPLC-UV system to characterize the pharmacokinetics of both nicotine and cotinine. Microdialysis probes were inserted into the jugular vein/right atrium and brain striatum of Sprague-Dawley rats, and nicotine (2 mg/kg, i.v.) was administered via the femoral vein. Dialysates were collected every 10 min and injected directly into a HPLC system. Both nicotine and cotinine were separated by a phenyl-hexyl column (150 mm x 4.6 mm) from dialysates within 12 min. The mobile phase consisted of an acetonitrile-methanol-20 mM monosodium phosphate buffer (55:45:900, v/v/v, pH adjusted to 5.1) with a flow-rate of 1 ml/min. The wavelength of the UV detector was set at 260 nm. The limit of quantification for nicotine and cotinine were 0.25 microg/ml and 0.05 microg/ml, respectively. Intra- and inter-day precision and accuracy of both measurements fell well within the predefined limits of acceptability. The blood and brain concentration-time profile of nicotine and cotinine suggests that nicotine is easily to get into the central nervous system and cotinine exhibits a long retention time and accumulates in blood.     

Improved gas chromatographic method for the determination of nicotine and cotinine in biologic fluids

Improved methods have been developed for the determination of nicotine and its major metabolite, cotinine, in blood, plasma, and urine samples. These methods utilize gas chromatography with alkali flame ionization (nitrogen--phosphorus) detection and structural analogs of nicotine and cotinine as internal standards.     

The determination of nicotine and cotinine in plasma

A method to determine plasma nicotine and cotinine simultaneously is described. After a simple extraction procedure, the amounts of nicotine and cotinine present in a sample are determined by gas chromatography using nitrogen-sensitive detection and a fused silica capillary column. The method has been demonstrated to detect nicotine and cotinine plasma concentrations within 1 and 9 ng/ml, respectively. The accuracy and precision of the method was demonstrated using spiked calf and human sera. Data are presented for the direct comparison of the present method with other methods of determination for plasma nicotine and cotinine.     

A validated method for the determination of nicotine, cotinine, trans-3'-hydroxycotinine, and norcotinine in human plasma using solid-phase extraction and liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry

A liquid chromatographic-mass spectrometric method for the simultaneous determination of nicotine, cotinine, trans-3'-hydroxycotinine, and norcotinine in human plasma was developed and validated. Analytes and deuterated internal standards were extracted from human plasma using solid-phase extraction and analyzed by liquid chromatography/atmospheric pressure chemical ionization-mass spectrometric detection with selected ion monitoring (SIM). Limits of detection and quantification were 1.0 and 2.5 ng/ml, respectively, for all analytes. Linearity ranged from 2.5 to 500 ng/ml of human plasma using a weighting factor of 1/x; correlation coefficients for the calibration curves were > 0.99. Intra- and inter-assay precision and accuracy were < 15.0%. Recoveries were 108.2-110.8% nicotine, 95.8-108.7% cotinine, 90.5-99.5% trans-3'-hydroxycotinine, and 99.5-109.5% norcotinine. The method was also partially validated in bovine serum, owing to the difficulty of obtaining nicotine-free human plasma for the preparation of calibrators and quality control (QC) samples. This method proved to be robust and accurate for the quantification of nicotine, cotinine, trans-3'-hydroxycotinine, and norcotinine in human plasma collected in clinical studies of acute nicotine effects on brain activity and on the development of neonates of maternal smokers.     

Simultaneous determination of plasma nicotine and cotinine by UHPLC–MS/MS in C57BL/6 mice and its application in a pharmacokinetic study

Plasma concentrations of nicotine and its active metabolite cotinine are highly correlated with its biological effects. A UHPLC–MS/MS method was developed, validated and applied for nicotine and cotinine analysis in mice plasma. Chromatographic separation was achieved on a BEH HILIC column using acetonitrile (0.1% formic acid) and 10 mm ammonium formate as mobile phase. The gradient elution was performed at 0.4 mL/min with a run time of 3.6 min. The quantitative ion transition was m/z 163.1 > 130.0 for nicotine, m/z 177.1 > 80.0 for cotinine and m/z 167.1 > 134.0 for nicotine‐D₄ (internal standard, IS). For both nicotine and cotinine, the calibration range was 5–500 ng/mL with 5 ng/mL as the lower limit of quantitation, and the intra‐ and inter‐day bias and imprecision were ?4.61–12.00% and <11.12%. The IS normalized recovery was 90.62–98.95% for nicotine and 89.18–101.53% for cotinine, and the IS normalized matrix factor was 106.00–116.44% for nicotine and 100.34–109.85% for cotinine. Both nicotine and cotinine were stable under conventional storage conditions. The validated method has been applied to a pharmacokinetic study in mice to calculate the pharmacokinetic parameters for both analytes.     

Gas chromatographic analysis of nicotine and cotinine in hair.

Non-invasive validation of cigarette- or cigar-smoking behaviour is necessary for large population studies. Urine or saliva samples can be used for confirmation of recent nicotine intake by analysis of cotinine, the major metabolite of nicotine. However, this test is not suitable for validation of survey data, since the quantification of cotinine in saliva only reflects nicotine exposure during the preceding week. To validate information on tobacco use, we investigated hair samples for quantifying nicotine and cotinine by gas chromatography-mass spectrometry. Hair (about 50-100 mg) was incubated in 1 M sodium hydroxide at 100 degrees C for 10 min. After cooling, samples were extracted by diethyl ether, using ketamine as an internal standard. Drugs were separated on a 12-m BP-5 capillary column, and detected using selected-ion monitoring (m/z 84, 98 and 180 for nicotine, cotinine and ketamine, respectively). Hair from non-smokers and smokers contained nicotine and cotinine. Although it is difficult to determine an absolute cut-off concentration, more than 2 ng of nicotine per milligram of hair can be used to differentiate smokers from non-smokers. Some applications of this technique are developed to determine the status of passive smokers, the gestational exposure in babies and the pattern of an individual's nicotine use by cutting strands of hair into sections of one-month intervals.     

Simultaneous quantification of oxysophocarpine and its active metabolite sophocarpine in rat plasma by liquid chromatography/mass spectrometry for a pharmacokinetic study

A sensitive, rapid and specific method for the simultaneous quantification of oxysophocarpine (OSC) and its active metabolite sophocarpine (SC) in rat plasma was developed and validated, using a liquid-liquid extraction procedure followed by liquid chromatography/electrospray ionization mass spectrometric (LC/ESI-MS) analysis. The separation was performed on a Zorbax Extend-C(18) column (2.1 mm i.d. x 50 mm, 5 microm) with a C(18) guard column using methanol-water containing 5 mm ammonium acetate (15:85, v/v) as mobile phase. Analysis was performed in selected ion monitoring (SIM) mode with an electrospray ionization (ESI) interface. [M + H](+) at m/z 263 for OSC, [M + H](+) at m/z 247 for SC and [M + H](+) at m/z 249 for matrine (internal standard) were selected as detecting ions, respectively. The method was linear in the concentration ranges 10-1000 ng/mL for OSC and 5-500 ng/mL for SC. The intra- and inter-day precisions (coefficient of variation) were within 7% for both analytes. Their accuracy (relative error) ranged from -6.4 to 1.5%. The limits of detection for OSC and SC were 3 and 1.5 ng/mL, respectively. The limits of quantitation for OSC and SC were 10 and 5 ng/mL, respectively. Recoveries of both analytes were greater than 85% at the low, medium and high concentrations. Both analytes were stable during all sample storage, preparation and analytic procedures. The method was successfully applied to a pharmacokinetic study after an oral administration of OSC to rats with a dose of 15 mg/kg.     

Determination of acetyltrimoprostil and its metabolite trimoprostil in human or dog plasma by gas chromatography-negative-ion chemical ionization mass spectrometry

A sensitive and specific procedure is described for the determination of the antisecretory prostaglandin acetyltrimoprostil and its metabolite trimoprostil in human or dog plasma using gas chromatography--negative-ion chemical ionization mass spectrometry (GC--NICI-MS). Trideuterated analogues of both compounds are added to plasma as the internal standards. The plasma is extracted at pH 7.3 with benzene--dichloromethane (9:1), and the residue of the organic extract is reacted at room temperature with pentafluorobenzyl bromide in the presence of 18-crown-6-ether and potassium acetate. The derivatives are reconstituted in heptane, and appropriate aliquots are analyzed by GC--NICI-MS with selected-ion monitoring of the intense (M--C6F5CH2)- fragment ions of acetyltrimoprostil (m/z 419), trimoprostil (m/z 377), and their respective trideuterated analogues (m/z 422 and m/z 380, respectively). Quantitation of an experimental plasma sample is based on a comparison of the m/z 419 versus m/z 422 and m/z 377 versus m/z 380 ion ratios in each sample to that obtained from the analysis of drug-free plasma fortified with various amounts of both protio compounds, and a fixed amount of each trideuterated internal standard. The limit of quantitation of the assay for human plasma is 0.2 ng ml-1 with mean relative standard deviations at this concentration of 15.5% and 9.7% for acetyltrimoprostil and trimoprostil, respectively.     

Hair analysis of nicotine and cotinine for evaluating tobacco smoke exposure by liquid chromatography-mass spectrometry

A simple liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method for the determination of nicotine and cotinine in human hair was established. In the procedure, a hair sample (10 mg) was washed with dichloromethane and digested in 2.5 M sodium hydroxide. The digest was extracted with dichloromethane and then 25 mM hydrochloric acid in methanol was added to the extract, to prevent loss of analytes. The solution was evaporated and redissolved in the mobile phase, methanol/10 mM ammonium acetate (30/70, v/v). A 20 microL aliquot of redissolved solution was subjected to analysis. Nicotine and cotinine in human hair were quantified by using deuterated analytes as internal standards. The quantification limits were 8 microg/L for nicotine and 0.9 microg/L for cotinine. The proposed method was applied to measure the concentrations of nicotine and cotinine in hair of smokers and non-smokers to evaluate their self-reported smoking and exposure to environmental tobacco smoke. In both cases, the method provided good selectivity, accuracy and precision.     

串联固相萃取-液相色谱-串联质谱法测定食品中嘧啶胺类杀菌剂残留

建立了食品中嘧啶胺类杀菌剂嘧霉胺、嘧菌胺及嘧菌环胺残留的串联固相萃取-液相色谱-串联质谱(HPLC-MS/MS)检测方法。胡萝卜、辣椒等样品经乙酸乙酯提取,石墨化炭黑-弗罗里硅藻土串联固相萃取柱(ENVI-Carb-Florisil SPE)净化后,在HPLC-MS/MS仪上进行检测分析,采用外标法定量。质谱分析采用电喷雾电离,正离子扫描,多反应监测模式。结果表明,柱净化后无明显的基质效应,嘧霉胺、嘧菌胺和嘧菌环胺在1～20 μg/L内相关系数可达0.9990以上,具有良好的线性关系;每种杀菌剂选择两个离子对,其中一组用于定量: 嘧霉胺m/z 200.1/107.1,嘧菌胺m/z 224.0/106.0及嘧菌环胺m/z 226.0/108.1;另一组用于确证: 嘧霉胺m/z 200.1/183.1,嘧菌胺m/z 224.0/131.1和嘧菌环胺m/z 226.0/133.1。样品中添加0.1、0.5、1.0 μg/kg的标准品,其回收率为73.2%～98.7%,相对标准偏差(n=10)小于10%;嘧霉胺、嘧菌胺、嘧菌环胺的检出限(信噪比(S/N)=3)均为0.03 μg/kg;嘧霉胺、嘧菌胺、嘧菌环胺的定量限(S/N=10)均为0.1 μg/kg。实验结果表明,该方法提取效果好,具有良好的灵敏度、回收率和重复性。     

Kinetic-energy-sensitive mass spectrometry for separation of different ions with the same m/z value

A double-focusing mass spectrometer (MS) equipped with a superconducting-tunnel-junction (STJ) detector has been applied to measure relative ionization cross-sections for the production of ions that are accompanied by different ion species with the same mass-to-charge (m/z) value. The STJ detector fabricated for this study enables kinetic energy (E) measurement of incoming individual ions at a counting rate of up to approximately 100 k ions/s and an energy resolution (DeltaE/E) of 15%. Both high counting rate and high-energy resolution are necessary to independently determine both m and z and not the m/z value only in ion-counting MS experiments. Ions such as (14)N(2) (2+) and (14)N(+) with the same m/z value can be clearly discriminated using a kinetic-energy-sensitive MS. This fine discrimination capability allows direct determination of relative ionization cross-sections of the homonuclear diatomic ions (14)N(2) (2+)/(14)N(2) (+) and (16)O(2) (2+)/(16)O(2) (+), which are difficult to measure due to the strong interference by the signals of their dissociated atomic ions with noticeably large ionization cross-sections. The new instrument requires no low-abundance heteronuclear diatomic molecules of the forms (14)N(15)N or (16)O(17)O to carry out ionization studies and thus, is expected to be useful in fields such as atmospheric science, interstellar science, or plasma physics.     

Detection and validated quantification of toxic alkaloids in human blood plasma--comparison of LC-APCI-MS with LC-ESI-MS/MS

Poisonings with toxic plants may occur after abuse, intentional or accidental ingestion of plants. For diagnosis of such poisonings, multianalyte procedures were developed for detection and validated quantification of the toxic alkaloids aconitine, atropine, colchicine, coniine, cytisine, nicotine and its metabolite cotinine, physostigmine, and scopolamine in plasma using LC-APCI-MS and LC-ESI-MS/MS. After mixed-mode solid-phase extraction of 1 ml of plasma, the analytes were separated using a C8 base select separation column and gradient elution (acetonitrile/ammonium formate, pH 3.5). Calibration curves were used for quantification with cotinine-d(3), benzoylecgonine-d(3), and trimipramine-d(3) as internal standards. The method was validated according to international guidelines. Both assays were selective for the tested compounds. No instability was observed after repeated freezing and thawing or in processed samples. The assays were linear for coniine, cytisine, nicotine and its metabolite cotinine, from 50 to 1000 ng/ml using LC-APCI-MS and 1 to 1000 ng/ml using LC-ESI-MS/MS, respectively, and for aconitine, atropine, colchicine, physostigmine, and scopolamine from 5 to 100 ng/ml for LC-APCI-MS and 0.1 to 100 ng/ml for LC-ESI-MS/MS, respectively. Accuracy ranged from -38.6 to 14.0%, repeatability from 2.5 to 13.5%, and intermediate precision from 4.8 to 13.5% using LC-APCI-MS and from -38.3 to 8.3% for accuracy, from 3.5 to 13.8%, for repeatability, and from 4.3 to 14.7% for intermediate precision using LC-ESI-MS/MS. The lower limit of quantification was fixed at the lowest calibrator in the linearity experiments. With the exception of the greater sensitivity and higher identification power, LC-ESI-MS/MS had no major advantages over LC-APCI-MS. Both presented assays were applicable for sensitive detection of all studied analytes and for accurate and precise quantification, with the exception of the rather volatile nicotine. The applicability of the assays was demonstrated by analysis of plasma samples from suspected poisoning cases.     

Profiling histidine-containing dipeptides in rat tissues by liquid chromatography/electrospray ionization tandem mass spectrometry

The histidine-containing dipeptides carnosine (CAR) and structurally related anserine (ANS) and homocarnosine (HCAR), widely distributed in vertebrate organisms, have recently been proposed as endogenous quenchers for highly cytotoxic alpha,beta-unsaturated aldehydes generated by peroxidation. A sensitive, selective, specific and rapid liquid chromatographic/electrospray ionization tandem mass spectrometric assay was developed and validated for the simultaneous determination of these peptides in biological matrices in order to establish their plasma/tissue distribution. Samples (plasma or tissue homogenates from male rats) were prepared by protein precipitation with HClO(4) (1 : 1, v/v) containing H-Tyr-His-OH as internal standard. The supernatant was separated on a Phenomenex Sinergy polar-RP column with a mobile phase of water-acetonitrile-heptafluorobutyric acid (9 : 1 : 0.01, v/v/v) at a flow-rate of 0.2 ml min(-1), with a run time of 10 min. Detection was effected on an ion trap mass spectrometer equipped with an electrospray ionization interface operating in positive ionization mode. The acquisitions were in the multiple reaction monitoring mode using the following precursor --> product ion combinations: H-Tyr-His-OH (internal standard) m/z 319 --> 301; CAR m/z 227 --> 210 + 209; ANS m/z 241 --> 224 + 197 + 170; HCAR m/z 241 --> 156. The method was validated over the concentration range 15-1000 nmol g(-1) and the limit of quantification (LOQ) and limit of detection (LOD) were 12.5 and 4.2 pmol injected, respectively. The intra- and inter-day precisions were <10% (< or =17.47% at the LOQ) and the intra- and inter-assay accuracies were within +/-10% for all concentrations. The mapping profile in rat tissue gave the following results: the highest concentrations of CAR and ANS were found in skeletal muscles (soleus, gastrocnemius, tibialis), followed by the heart, cerebellum and brain (ANS below the LOQ). HCAR was found only in the brain and cerebellum. No histidine-containing dipeptides were detectable in plasma, liver, kidney and lung.     

Sensitive and rapid method for the determination of urinary cotinine in non-smokers: an application for studies assessing exposures to second hand smoke (SHS)

We describe a sensitive and rapid method to assay urinary cotinine levels among non-smokers using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) and its application in studies assessing exposures to second hand smoke (SHS). Cotinine was initially extracted from 1 ml of urine with methylene chloride by using a liquid-liquid extraction Chem Elut™ column. The extracted sample was further separated by using a BetaBasic C18 column (1 mm × 150 mm, 3 μm) with isocratic elution (60:40 acetonitrile and 5 mM ammonium acetate at pH 5), and then examined using a triple quadrupole mass spectrometer with an electrospray ionization (ESI) source in multiple-reaction-monitoring (MRM) mode. The elution of cotinine from the LC column took approximately 2.3 min and the detection of cotinine by ESI/MS/MS provided a limit of detection (LOD) of 0.3 ng/ml. The ESI/MS/MS detection was able to easily distinguish between cotinine and nicotine. This method, validated using a cotinine concentration range from 0.8 to 102.4 ng/ml, was successfully applied in a cross-sectional study examining differences in levels and sources of second hand smoke (SHS) exposure among non-smokers. Self-reported measures of SHS exposure were significantly associated with urinary cotinine levels. This urinary cotinine assay using LC-ESI/MS/MS provides a robust, high throughput and very sensitive method for the evaluation of SHS exposure for use in epidemiologic and clinical research studies.     

Analysis of testosterone and dihydrotestosterone from biological fluids as the oxime derivatives using high-performance liquid chromatography/tandem mass spectrometry

A sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method for the simultaneous quantitative analysis of dihydrotestosterone (DHT) and testosterone (T) from biological fluids has been developed. Commercially available deuterated analogues were used as internal standards. Steroids were extracted from serum or testicular fluid with hexane/ethyl acetate, evaporated to dryness, and treated with hydroxylamine to form their oxime derivatives. Upon chromatographic separation, the compounds were quantified using selected reaction monitoring (SRM). For T, the [M+H](+) ion at m/z 304 and the fragment ion at m/z 124 were used as the precursor and product ions. For DHT the ion cluster [M+H+ACN](+) at m/z 347 and the dissociated ion [M+H](+) at m/z 306 were used as the precursor and product ions, respectively. The limits of detectability on-column were in the sub-femtomole range for both compounds and the intra-day coefficient of variation (CV) for analysis from serum was less than 7% for both compounds. Given its high reproducibility, sensitivity, and relative simplicity, this assay should be of use in determining androgen levels in biospecimens, particularly in settings where sample quantity or steroid concentration are low.