Biological variation in clozapine and metabolite reporting during therapeutic drug monitoring.

BACKGROUND: Clozapine (CLO) is an atypical antipsychotic used in management of treatment-resistant schizophrenia. Adverse drug reactions are caused by both CLO and its primary metabolite, norclozapine (NCLO). We defined the biological variability of CLO, NCLO, and the CLO to NCLO ratio (CNR) as well as assess the impact of reporting CLO and NCLO routinely. METHODS: The CVi and CVg were calculated from 1904 results from 247 patients by CV-ANOVA, and ANOVA, respectively, for CLO, NCLO, and the CNR. Association between each were also analyzed against a number of parameters including age and gender, complete blood count (CBC), kidney and liver function tests, lipids, and glucose within 24 h of CLO measurement. RESULTS: For CLO, NCLO and CNR, the CVi was calculated as 19.3%, 19.2%, and 14.7%, and the CVg was 46.9%, 51.4%, and 36.3%, respectively. A total of 87 patients (19.7%) demonstrated higher NCLO results than CLO, with a ratio as low as 0.47. Kidney function was also found to have a statistically significant relationship to CLO and NCLO levels. CONCLUSIONS: We provide data for biological variability of CLO metabolism as well as while providing some evidence for reporting NCLO values clinically.

Updated reference intervals for urine normetanephrine have no effect on test sensitivity but fewer false positives.

OBJECTIVE: We previously highlighted the problem of frequent false positives in 24 h urine normetanephrine(UNM) measurements owing to reference intervals that are inappropriately low for the population being screened for pheochromocytoma. Using a large population database, we devised new age-stratified reference intervals for the 24 h UNM test that were higher compared to previous. However, it was uncertain as to whether this would compromise test sensitivity for true pheochromocytoma cases. DESIGN AND METHODS: Retrospective analysis of all pheochromocytoma cases from a recently constructed provincial registry. All confirmed cases had their diagnostic UNM results retrospectively re-analysed according to the newly proposed UNM reference intervals to determine the percentage and phenotype of cases that might have been theoretically missed with the new reference range. RESULTS: After excluding pediatric and non-secretory head and neck paragangliomas, there were 60 confirmed pheochromocytoma cases. Using prior reference intervals, 51/60 (85%) had an abnormally high UNM. Of the 9 with normal UNM, 4 had a high urine metanephrine(UMN), 5 had normal levels of both UNM and UMN such that 55/60 had abnormal test results, representing the historical combined test sensitivity of 92%. Using the proposed reference interval, 43/60 (72%) had high UNM results. Of the 17 with normal UNM, 12 had high UMN, 5 had normal levels of both UNM and UMN. Therefore, 55/60 patients had had elevations in either UNM or UMN, corresponding to an identical combined test sensitivity of 92%. CONCLUSIONS: Reference intervals for UNM derived from actual clinical population screening data are higher than in traditional healthy volunteers. Use of these more appropriate reference intervals can significantly reduce the false positive rate without compromising test sensitivity for true pheochromocytoma.

Inpatient Measurements of Urine Metanephrines are Indistinguishable from Pheochromocytoma: Retrospective Cohort Study.

BACKGROUND: Pheochromocytoma is a rare cause of acute cardiovascular disease; however, any severe illness may have high catecholamines, simulating pheochromocytoma. We determined the spectrum of urine metanephrines from inpatient and outpatient collections without pheochromocytoma, compared with confirmed pheochromocytoma patients. METHODS: Retrospective analysis using centralized laboratory data serving all outpatients and hospitals in southern Alberta. The analysis comprised 24-hour urine normetanephrine and metanephrine (UNM-UMN) results collected from hospital inpatients, community outpatients, and patients from a comprehensive provincial pheochromocytoma registry. RESULTS: There were 974 unique inpatients (including 132 from intensive care), 6802 outpatients, and 58 pheochromocytoma patients. Among outpatient, general ward, and intensive care unit (ICU) patients, 18.7%, 34.4%, and 67.4% of results, respectively, were supranormal. Although pheochromocytoma patients had higher median UNM-UMN vs inpatients, there was substantial overlap. Receiver operating characteristic (ROC) analysis showed area under the curve (AUC) of 0.64-0.91 to detect true pheochromocytoma (P < .0001), with progressively poorer discrimination among hospitalized and ICU-dependent patients. A 24-hour urine normetanephrine >6.95 nmol/d had 98% specificity for pheochromocytoma when inpatient general ward samples were included, but only 46% sensitivity and 13% positive predictive value for pheochromocytoma. Considering ICU collections, 98% specificity required results more than fivefold above the upper reference limit and still had poor positive predictive value. A model combining both UNM and UMN results as a cross-product marginally improved the ROC AUC, but improved sensitivity in outpatients and ward patients but not ICU patients. CONCLUSION: There is a high degree of overlap in UNM-UMN between hospitalized patients and pheochromocytoma; high test specificity is not achieved in this population unless >3-5 times the upper reference limit.

Properly Collected Plasma Metanephrines Excludes PPGL After False-Positive Screening Tests.

CONTEXT: False-positive results are common for pheochromocytoma/paraganglioma (PPGL) real-world screening. OBJECTIVE: Determine the correlation between screening urine and seated plasma metanephrines in outpatients where PPGL was absent, compared to meticulously prepared and supine-collected plasma metanephrines with age-adjusted references. DESIGN: Retrospective cohort study. SETTING: Databases from a single-provider provincial laboratory (2012-2018), a validated PPGL registry, and a manual chart review from a specialized endocrine testing unit. PATIENTS: PPGL registry data excluded known PPGL cases from the laboratory database. Outpatients having both urine and plasma metanephrines <90 days apart. METHODS: The correlation between urine and seated plasma measures along with the total positivity rate. All cases of plasma metanephrines drawn in the endocrine unit were reviewed for test indication and test positivity rate. RESULTS: There were 810 non-PPGL pairs of urine and plasma metanephrines in the laboratory database; 46.1% of urine metanephrines were reported high. Of seated outpatient plasma metanephrines drawn a median of 5.9 days later, 19.2% were also high (r = 0.33 and 0.50 for normetanephrine and metanephrine, respectively). In contrast, the meticulously prepared and supine collected patients (n = 139, 51% prior high urine metanephrines) had <3% rate of abnormal high results in patients without known PPGL/adrenal mass. CONCLUSIONS: There was a poor-to-moderate correlation between urine and seated plasma metanephrines. Up to 20% of those with high urine measures also had high seated plasma metanephrines in the absence of PPGL. Properly prepared and collected supine plasma metanephrines had a false-positive rate of <3% in the absence of known PPGL/adrenal mass.

Epidemiology of pheochromocytoma and paraganglioma: population-based cohort study.

OBJECTIVE: Despite the significant morbidity and mortality associated with pheochromocytoma and paraganglioma, little is known about their epidemiology. The primary objective was to determine the incidence of pheochromocytoma and paraganglioma in an ethnically diverse population. A secondary objective was to develop and validate algorithms for case detection using laboratory and administrative data. DESIGN: Population-based cohort study in Alberta, Canada from 2012 to 2019. METHODS: Patients with pheochromocytoma or paraganglioma were identified using linked administrative databases and clinical records. Annual incidence rates per 100 000 people were calculated and stratified according to age and sex. Algorithms to identify pheochromocytoma and paraganglioma, based on laboratory and administrative data, were evaluated. RESULTS: A total of 239 patients with pheochromocytoma or paraganglioma (collectively with 251 tumors) were identified from a population of 5 196 368 people over a period of 7 years. The overall incidence of pheochromocytoma or paraganglioma was 0.66 cases per 100 000 people per year. The frequency of pheochromocytoma and paraganglioma increased with age and was highest in individuals aged 60-79 years (8.85 and 14.68 cases per 100 000 people per year for males and females, respectively). An algorithm based on laboratory data (metanephrine >two-fold or normetanephrine >three-fold higher than the upper limit of normal) closely approximated the true frequency of pheochromocytoma and paraganglioma with an estimated incidence of 0.54 cases per 100 000 people per year. CONSLUSION: The incidence of pheochromocytoma and paraganglioma in an unselected population of western Canada was unexpectedly higher than rates reported from other areas of the world.

Moderate renal impairment does not preclude the accuracy of 24-hour urine normetanephrine measurements for suspected pheochromoctyoma.

OBJECTIVE: A 24-hour urine nor/metanephrine (urine NM-MN) measurements are a recommended first step in pheochromocytoma diagnosis. We hypothesized the presence of renal impairment (CKD) significantly confounds the results obtained in a urine NM-MN collection, giving artificially lower measurements. DESIGN: Retrospective review of a comprehensive laboratory database with all urine NM-MN results from Southern Alberta from 2010 to 2018 (n = 15 505). After excluding high probability pheochromocytoma cases, results from patients with three levels of CKD (n = 796) were compared to those without CKD to determine the potential CKD effect. PATIENTS: All patients having urine NM-MN collection during the time period, irrespective of ordering physician or test indication. MEASUREMENTS: Urine NM-MN was measured by liquid chromatography-tandem mass spectrometry and glomerular filtration rate determined within a median of 1.9 days, as estimated by CKD-EPI equation. RESULTS: In subjects with mild-to-moderate renal impairment, there was no continuous gradient between subnormal renal function and urine NM-MN measures. When the estimated GFR was < 15 mL/min/m2 , the hypothesized effect on lowered urine NM-MN became apparent. CONCLUSIONS: A 24-hour urine NM-MN measurement is unlikely to be affected by mild-to-moderate renal impairment and may be used as a reliable diagnostic test. With more advanced renal impairment, CKD-specific reference ranges or an alternative test may be needed.

A high rate of modestly elevated plasma normetanephrine in a population referred for suspected PPGL when measured in a seated position.

OBJECTIVE: Determine rate of high plasma normetanephrine or metanephrine (PNM-PMN) in a large sample of patients according to PNM-PMN posture and age-adjusted references. DESIGN: Retrospective re-analysis of PNM-PMN from a Canadian reference laboratory (n = 5452), 2011-2015; most were in seated position (n = 5112) rather than supine (n = 340). An international PPGL database demonstrated expected distribution of supine PNM-PMN in PPGL patients. METHODS: All PNM-PMN from a tertiary referral laboratory were reviewed. Any PNM-PMN result greater than 2× upper reference limit (URL) was considered likely true PPGL. Results 1-2× URL were uncertain, requiring additional testing/follow-up despite most being false positive given the rarity of PPGL. The rate of results in the 1-2× URL category were calculated for each group according to collection posture and differing published URL: seated, supine or supine age adjusted. RESULTS: When collected and interpreted by seated URL, 19.6% of PNM required additional testing; only 4.6% being >2× URL. For patients over age 50 years, the abnormal rate was 24.9%. When collected supine, interpreted by supine age-adjusted URL, only 5.3% of PNM were mildly elevated. Possible false positives may be even lower when considering PMN or plasma methoxytyramine which were commonly high in true PPGL despite mild PNM elevations. CONCLUSIONS: In a general medical population, seated PNM has a high rate of abnormal results, far exceeding expected prevalence. Supine measurement with supine, age-adjusted interpretation is strongly preferred prior to costly or invasive PPGL investigations. SUMMARY: Review of 5452 plasma normetanephrine measurements showed 20% to be high, likely false positives for most. Supine, age-adjusted measures were half as likely to be elevated.

Concordance between laboratories in metal ion testing in patients with metal-on-metal hip implants.

BACKGROUND: Testing of whole blood or serum metal ion levels has become an important part of assessing and monitoring the performance of metal-on-metal bearings, both in hip resurfacing arthroplasty and in total hip replacement. The aim of this study was to determine the concordance between 2 laboratories testing cobalt and chromium ion levels in patients with metal-on-metal bearings. METHODS: Serum and whole blood samples from patients who had undergone metal-on-metal resurfacing or large-diameter total hip arthroplasty were tested for cobalt and chromium ions in laboratory A (a recognized laboratory) and laboratory B (tasked with testing clinical specimens). Laboratory A performed cobalt and chromium testing on whole blood, and laboratory B performed cobalt testing on whole blood and chromium testing on serum. RESULTS: Samples from 104 patients were tested. Laboratory B reported lower whole blood cobalt levels than laboratory A. Furthermore, laboratory A reported that all patients had elevated whole blood cobalt ion levels compared to the normal reference values for the laboratory, whereas laboratory B reported that 46 patients (44.2%) had whole blood cobalt ion levels within the normal reference range for the laboratory. CONCLUSION: This comparative study highlights the importance of using a single laboratory for metal ion testing, as values generated from different laboratories may not be directly comparable. With recent literature suggesting that whole blood cobalt levels as low as 1 ppb may be a predictor of adverse reactions to metal debris, accurate clinical measurement needs to be increasingly exact.

CONTEXTE: Le dosage sanguin ou sérique d'ions métalliques est devenu une étape importante de l'évaluation et du suivi des prothèses à couple de frottement métal-métal utilisées en arthroplastie de resurfaçage ou totale de la hanche. La présente étude visait à évaluer la concordance entre les résultats de 2 laboratoires pour le dosage du cobalt et du chrome chez des patients porteurs de ces prothèses. MÉTHODES: Des prélèvements de sérum et de sang entier de patients porteurs d'une prothèse de resurfaçage ou d'une prothèse totale à grand diamètre de hanche à couple métal-métal ont été expédiés au laboratoire A (un laboratoire reconnu) et au laboratoire B (spécialisé en analyse d'échantillons cliniques) pour le dosage des ions cobalt et chrome. Le laboratoire A a effectué toutes ses analyses sur des prélèvements de sang entier, et le laboratoire B a utilisé le sang entier pour le dosage du cobalt et le sérum pour le dosage du chrome. RÉSULTATS: Les prélèvements de 104 patients ont été analysés. Le laboratoire B a détecté des taux sanguins de cobalt inférieurs à ceux du laboratoire A. De plus, le laboratoire A a indiqué que tous les patients présentaient des taux de cobalt sanguins élevés par rapport à ses valeurs de référence, alors que le laboratoire B a déterminé que le taux de cobalt sanguin de 46 patients (44,2 %) se trouvait dans sa fourchette de valeurs de référence normales. CONCLUSION: Cette étude comparative vient souligner l'importance de choisir un seul laboratoire pour le dosage des ions métalliques, car les valeurs générées par des établissements différents pourraient ne pas être directement comparables. Comme des études récentes semblent indiquer que des taux de cobalt sanguins aussi faibles que 1 p. p. milliard pourraient être des prédicteurs de réaction indésirable aux débris métalliques, la précision et l'exactitude des mesures cliniques revêtent une importance croissante.

One-step extraction and quantitation of toxic alcohols and ethylene glycol in plasma by capillary gas chromatography (GC) with flame ionization detection (FID).

OBJECTIVES: Clinical analysis of volatile alcohols (i.e. methanol, ethanol, isopropanol, and metabolite acetone) and ethylene glycol (EG) generally employs separate gas chromatography (GC) methods for analysis. Here, a method for combined analysis of volatile alcohols and EG is described. DESIGN AND METHODS: Volatile alcohols and EG were extracted with 2:1 (v:v) acetonitrile containing internal standards (IS) 1,2 butanediol (for EG) and n-propanol (for alcohols). Samples were analyzed on an Agilent 6890 GC FID. The method was evaluated for precision, accuracy, reproducibility, linearity, selectivity and limit of quantitation (LOQ), followed by correlation to existing GC methods using patient samples, Bio-Rad QC, and in-house prepared QC material. RESULTS: Inter-day precision was from 6.5-11.3% CV, and linearity was verified from down to 0.6mmol/L up to 150mmol/L for each analyte. The method showed good recovery (~100%) and the LOQ was calculated to be between 0.25 and 0.44mmol/L. Patient correlation against current GC methods showed good agreement (slopes from 1.03-1.12, and y-intercepts from 0 to 0.85mmol/L; R(2)>0.98; N=35). Carryover was negligible for volatile alcohols in the measuring range, and of the potential interferences tested, only toluene and 1,3 propanediol interfered. The method was able to resolve 2,3 butanediol, diethylene glycol, and propylene glycol in addition to the peaks quantified. CONCLUSIONS: Here we describe a simple procedure for simultaneous analysis of EG and volatile alcohols that comes at low cost and with a simple liquid-liquid extraction requiring no derivitization to obtain adequate sensitivity for clinical specimens.

Identification of tobacco-specific nitrosamines as disinfection byproducts in chloraminated water.

Tobacco-specific nitrosamines (TSNAs) exist in environmental waters; however, it is unknown whether TSNAs can be produced during water disinfection. Here we report on the investigation and evidence of TSNAs as a new class of disinfection byproducts (DBPs). Using five common TSNAs, including (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) as the targets, we first developed a solid phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) method capable of rapidly determining these TSNAs at levels as low as 0.02 ng/L in treated water. Using this highly sensitive method, we investigated the occurrence and formation potential (FP) (precursor test conducted in the presence of chloramines) of TSNAs in treated water from two wastewater treatment plants (WWTPs) and seven drinking water treatment plants (DWTPs). NNAL was detected in the FP samples, but not in the samples before the FP test, confirming NNAL as a DBP. NNK was detected in the treated wastewater before the FP test, but its concentration increased significantly after chloramination in two of three tests. Thus, NNK could be a DBP and/or a contaminant in wastewater. Moreover, these TSNAs were detected in FP tests of wastewater-impacted DWTP plant influents in 9 of 11 samples. However, TSNAs were not detected at full-scale DWTPs, except for at one DWTP with high ammonia where breakpoint chlorination was not achieved. The concentration of the sum of five TSNAs (0.3 ng/L) was 100-fold lower than NDMA, suggesting that TSNAs have a minor contribution to total nitrosamines in water. We examined several factors in the treatment process and found that chlorine or ozone may destroy TSNA precursors and granular activated carbon (GAC) treatment may remove the precursors. Further research is warranted into the efficiency of these processes at different DWTPs using sources of varying water quality.

Liquid chromatography tandem mass spectrometry determination of free and conjugated estrogens in breast cancer patients before and after exemestane treatment.

We report liquid chromatographic separation with tandem mass spectrometry determination of 12 endogenous estrogens and their intact conjugates in blood and urine and its application to study effects of exemestane treatment on estrogen generation and metabolism in postmenopausal women with estrogen-dependent breast cancer. A 0.5 mL aliquot of each urine or serum sample is fractionated with solid phase extraction to a fraction of free estrogen and another fraction of their conjugates. The reversed phase LC/MS/MS determines dansylated estrogens with positive ionization and intact conjugates with negative ionization. The method provides reproducible separation and limit of detection as low as 1 pg mL(-1) for free estrogens and 0.03 ng mg(-1) creatinine for the conjugates in serum and urine samples. The method enabled us to acquire unique concentration profiles of 12 endogenous estrogens and their intact conjugates in 30 breast cancer patients before and after one month of exemestane treatment. Exemestane suppressed total serum and urinary estrogens by 11-97% (P<0.0001) and 8.7-91% (P<0.0001), respectively. Specifically, these data show that exemestane preferentially suppressed E1, E1-3S, E1-3G, and E2-17G more than other estrogens. Linear regression analysis of estrogen concentrations before and after treatment showed correlation coefficients of 0.8385 (n=289, P<0.0001) and 0.8863 (n=360, P<0.0001). This study provides urinary and blood estrogen concentration profiles in breast cancer patients to demonstrate the effect of exemestane on estrogen generation, supporting inhibition of aromatase activity.

Characterization and determination of chloro- and bromo-benzoquinones as new chlorination disinfection byproducts in drinking water.

We report the characterization and determination of 2,6-dichloro-1,4-benzoquinone and three new disinfection byproducts (DBPs): 2,6-dichloro-3-methyl-1,4-benzoquinone, 2,3,6-trichloro-1,4-benzoquinone, and 2,6-dibromo-1,4-benzoquinone. These haloquinones are suspected bladder carcinogens and are likely produced during drinking water disinfection treatment. However, detection of these haloquinones is challenging, and consequently, they have not been characterized as DBPs until recently. We have developed an electrospray ionization tandem mass spectrometry technique based on our observation of unique ionization processes. These chloro- and bromo-quinones were ionized through a reduction step to form [M + H](-) under negative electrospray ionization. Tandem mass spectra and accurate mass measurements of these compounds showed major product ions, [M + H - HX](-), [M + H - HX - CO](-), [M + H - CO](-), and/or X(-) (where X represents Cl or Br). The addition of 0.25% formic acid to water samples was found to effectively stabilize the haloquinones in water and to improve the ionization for analysis. These improvements were rationalized from the estimates of pK(a) values (5.8-6.3) of these haloquinones. The method of tandem mass spectrometry detection, combined with sample preservation, solid phase extraction, and liquid chromatography separation, enabled the detection of haloquinones in chlorinated water samples collected from a drinking water treatment plant. The four haloquinones were detected only in drinking water after chlorination treatment, with concentrations ranging from 0.5 to 165 ng/L, but were not detectable in the untreated water. This method will be useful for future studies of occurrence, formation pathways, toxicity, and control of these new halogenated DBPs.

Elevated urinary leukotriene E4 levels are associated with hospitalization for pain in children with sickle cell disease.

Cysteinyl leukotrienes (CsyLTs) are inflammatory mediators produced by white blood cells. Leukotriene LTE(4) is the stable metabolite of CsyLTs, which can be measured in urine. We tested two hypotheses among children with sickle cell disease (SCD): (1) baseline urinary LTE(4) levels are elevated in children with SCD when compared with controls; and (2) baseline LTE(4) levels are associated with an increased incidence rate of hospitalization for SCD-related pain. Baseline LTE(4) levels were measured in children with SCD (cases) and children without SCD matched for age and ethnicity (controls). Medical records of cases were reviewed to assess the frequency of hospitalization for pain within 3 years of study entry. LTE(4) levels were obtained in 71 cases and 22 controls. LTE(4) levels were higher in cases compared with controls (median LTE(4): 100 vs. 57 pg/mg creatinine, P < 0.001). After adjustment for age and asthma diagnosis, a greater incidence rate of hospitalization for pain was observed among children with SCD in the highest LTE(4) tertile when compared with the lowest (114 vs. 52 episodes per 100 patient-years, P = 0.038). LTE(4) levels are elevated in children with SCD when compared with controls. LTE(4) levels are associated with an increased rate of hospitalizations for pain.

A cell-microelectronic sensing technique for profiling cytotoxicity of chemicals.

A cell-microelectronic sensing technique is developed for profiling chemical cytotoxicity and is used to study different cytotoxic effects of the same class chemicals using nitrosamines as examples. This technique uses three human cell lines (T24 bladder, HepG2 liver, and A549 lung carcinoma cells) and Chinese hamster ovary (CHO-K1) cells in parallel as the living components of the sensors of a real-time cell electronic sensing (RT-CES) method for dynamic monitoring of chemical toxicity. The RT-CES technique measures changes in the impedance of individual microelectronic wells that is correlated linearly with changes in cell numbers during t log phase of cell growth, thus allowing determination of cytotoxicity. Four nitrosamines, N-nitrosodimethylamine (NDMA), N-nitrosodiphenylamine (NDPhA), N-nitrosopiperidine (NPip), and N-nitrosopyrrolidine (NPyr), were examined and unique cytotoxicity profiles were detected for each nitrosamine. In vitro cytotoxicity values (IC(50)) for NDPhA (ranging from 0.6 to 1.9 mM) were significantly lower than the IC(50) values for the well-known carcinogen NDMA (15-95 mM) in all four cell lines. T24 cells were the most sensitive to nitrosamine exposure among the four cell lines tested (T24>CHO>A549>HepG2), suggesting that T24 may serve as a new sensitive model for cytotoxicity screening. Cell staining results confirmed that administration of the IC(50) concentration from the RT-CES experiments inhibited cell growth by 50% compared to the controls, indicating that the RT-CES method provides reliable measures of IC(50). Staining and cell-cycle analysis confirmed that NDPhA caused cell-cycle arrest at the G0/G1 phase, whereas NDMA did not disrupt the cell cycle but induced cell death, thus explaining the different cytotoxicity profiles detected by the RT-CES method. The parallel cytotoxicity profiling of nitrosamines on the four cell lines by the RT-CES method led to the discovery of the unique cytotoxicity of NDPhA causing cell-cycle arrest. This study demonstrates a new approach to comprehensive testing of chemical toxicity.

Cell-electronic sensing of particle-induced cellular responses.

We report a new technique for the continuous and real-time measurement of microparticle-induced cellular responses using a real-time cell-electronic sensing (RT-CES) technology. The method involves the use of microelectrode-embedded microwells seeded with one of two lung cancer carcinoma cell lines (A549 and SK-MES-1), allowing for continuous measurements of impedance. The change in impedance that is automatically converted to the cell index is linearly correlated with the numbers of the seeding cells during the log phase, providing quantitative measurement of cytotoxicity. After 24 h of initial incubation in 96 microwells, the cultures are treated with microparticles, and changes in the cell index are monitored in real time. Multiple data, including dose response curves, IC(50) (a concentration inhibiting 50% cell growth), and cell-specific and particulate-specific cell responses, are obtained from a single set of experiments. SK-MES-1 cells consistently showed more severe effects and lower IC(50) values than A549 cells when they were treated with quartz particle suspensions. The different effects detected using the RT-CES technique were related to morphological change and apoptosis, supported by the scanning electronic microscopy and flow cytometry results. The method is further used to test the cytotoxicity of two PM(10) standard reference materials of urban air dust and diesel particulates, demonstrating the potential application of this new technique for biomonitoring of air particulates.

Characterization of new nitrosamines in drinking water using liquid chromatography tandem mass spectrometry.

N-Nitrosodimethylamine (NDMA), a member of a group of probable human carcinogens, has been detected as a disinfection byproduct (DBP) in drinking water supplies in Canada and the United States. To comprehensively investigate the occurrence of possible nitrosamines in drinking water supplies, a liquid chromatography-tandem mass spectrometry technique was developed to detect both thermally stable and unstable nitrosamines. This technique consisted of solid-phase extraction (SPE), liquid chromatography (LC) separation, and tandem quadrupole linear ion trap mass spectrometry (MS/MS) detection. It enabled the determination of sub-ng/L levels of nine nitrosamines. Isotope-labeled N-nitrosodimethylamine-d6 (NDMA-d6) was used as the surrogate standard for determining recovery, and N-nitrosodi-n-propylamine-dl4 (NDPA-dl4) was used as the internal standard for quantification. The method detection limits were estimated to be 0.1-10.6 ng/L, and the average recoveries were 41-111% for the nine nitrosamines; of these, NDMA, N-nitrosopyrrolidine (NPyr), N-nitrosopiperidine (NPip), and N-nitrosodiphenylamine (NDPhA) were identified and quantified in drinking water samples collected from four locations within the same distribution system. In general, the concentrations of these four nitrosamines in this distribution system increased with increasing distance from the water treatment plant, indicating that the amount of formation was greater than the amount of decomposition within this time frame. The identification of NPip and NDPhA in drinking water systems and the distribution profiles of these nitrosamines have not been reported previously. These nitrosamines are toxic, and their presence as DBPs in drinking water may have toxicological relevance.