Determination of Barbiturates by Femtosecond Ionization Mass Spectrometry.

Barbiturates are highly susceptible to dissociation in mass spectrometry (MS) because of their long side chains combined with a nonaromatic ring consisting of several carbonyl and amine groups. As a result, they exhibit extensive α-cleavage and subsequent rearrangement, making the identification of these compounds difficult. Although a library of electron ionization MS (EIMS) is available, most barbiturates have very similar fragment patterns. Accordingly, it would be desirable to develop a technique for soft ionization, providing a molecular ion and large fragment ions as well. In this study, a molecular ion was clearly observed, in addition to large fragment ions, for a variety of barbiturates based on multiphoton ionization MS (MPIMS) using a tunable ultraviolet femtosecond laser as the ionization source (fs-LIMS). This favorable result was achieved when the optimal laser wavelength for minimizing the excess energy remaining in the ionic state was used. An examination of the photofragmentation pathways suggested that an H atom in the side chain was abstracted by an oxygen atom in the carbonyl group in the ring structure thus initiating fragmentation and subsequent rearrangement. Barbiturates that are substituted with alkyl groups (amobarbital and pentobarbital) had narrower spectral regions for optimal ionization than the other barbiturates with alkyl and alkenyl groups (butalbital and secobarbital) and more with alkyl and phenyl groups (phenobarbital). All of the barbiturates studied provided unique mass spectral patterns in fs-LIMS, which was useful for the reliable identification of these compounds in practical trace analysis.

Quantitative analysis of thiamylal and its metabolite secobarbital using liquid chromatography-tandem mass spectrometry in adipose tissue, serum, and liver.

Thiamylal is an ultrashort-acting barbiturate used for intravenous administration or general anesthesia induction. However, some cases of poisoning and suicide with thiamylal administration have been reported. Additionally, there are few reports on its analysis in the organs and adipose tissue, which requires purification by column chromatography and evaporation. A rapid and sensitive method was developed for quantifying thiamylal and its metabolite, secobarbital, in the adipose tissue, serum, and liver using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Samples were prepared using modified QuEChERS extraction. For adipose tissue samples, an acetonitrile-hexane partitioning step was added to the extraction. This method was applied to investigate a suspected self-poisoning autopsy case. The quantitation accuracy for thiamylal added to porcine pericardial fat (0.18 µg/g), human serum (0.015 µg/mL), and porcine liver (0.18 µg/g) was 103%, 113%, and 95.3%, respectively. The quantitation limits calculated for porcine pericardial fat, human serum, and porcine liver at a signal-to-noise ratio of 10 were 0.06 µg/g, 0.005 µg/mL, and 0.06 µg/g, respectively. In addition, the thiamylal and secobarbital levels in the forensic autopsy case were 140 and 1.5 µg/g, respectively, in myocardial fat; 3.5-4.9 and 0.12-0.20 µg/mL, respectively, in serum; and 6.2-42 and 0.58-1.1 µg/g, respectively, in liver tissue. Thiamylal is especially distributed in the adipose tissue. The thiamylal-to-fat ratio may help estimate the time from administration to death. The developed modified QuEChERS extraction method with acetonitrile-hexane partitioning is suitable for analyzing hydrophobic compounds, such as thiamylal, in the adipose tissue.

High-throughput determination of valproate in human samples by modified QuEChERS extraction and GC-MS/MS.

A new high-throughput method was developed for analysis of valproate in human plasma samples by QuEChERS extraction and gas chromatography-tandem mass spectrometry (GC-MS/MS). Plasma samples (0.2 ml) spiked with valproate and secobarbital-d5 (internal standard) were diluted with 1.3 ml of distilled water. Acetonitrile (1 ml) was added followed by 0.4 g MgSO4 and 0.1 g NaOAC. After a centrifugation step (2000 g for 10 min), 1 ml of the supernatant was transferred to a dispersive-solid phase extraction (dSPE) tube containing 150 mg MgSO4 and 50 mg C18. This mixture was vortexed and centrifuged at 3000 g for 5 min, and then the upper layer was evaporated to dryness under a stream of nitrogen. The residue was dissolved in 40 µl ethyl acetate, and a 1-µl aliquot was injected into the GC-MS/MS. The GC separation of the compounds was achieved on a fused-silica capillary column Rxi-5Sil MS (30 m × 0.25 mm i.d.; 0.25-µm film thickness) and detected by MS/MS operating in electron ionization ion source mode. The regression equations showed excellent linearity (r > 0.9997) from 50 to 5000 ng/ml for plasma, with limit of detection of 10 ng/ml. The extraction efficiency of valproate for plasma ranged between 71.2%-103.5%. The coefficient of variation was <18.5%. The method was successfully applied to actual analyses of an autopsy case. This method can be useful for simple and reliable measurements of valproate in clinical and toxicological analyses; it can be integrated in screening and simultaneous determination methods for multiple drugs and poisons in the further studies.

[Clinical observation on fire needles at bones combined with cupping and Tuina for knee osteoarthritistis].

OBJECTIVE: To explore a better therapy for knee osteoarthritis. METHODS: One hundred cases were randomly divided into a comprehensive group and an acupuncture group, 50 cases in each one. The comprehensive treatment of fire needles at bones combined with cupping and Tuina on local area of affected knee was applied in the comprehensive group. The Ashi points were mainly selected in the fire needles at bones therapy, once every other day. The cupping and Tuina therapy was adopted once a day. The conventional acupuncture was applied in the acupuncture group, in which Dubi (ST 35), Neixiyan (EX-LE 4), Xuehai (SP 10), Liangqiu (ST 34) and so on were selected, once a day. Ten days of treatment were taken as a treatment course in both two groups, and totally 1 to 2 courses was required. The pain score of joint before and after the treatment was observed and efficacy was assessed in two groups. RESULTS: Compared before the treatment, the pain score of joint after the treatment was obviously improved in two groups (both P<0.05), and the score in the comprehensive group was lower than that in the acupuncture group (P<0.05). The clinical cured rate was 38.0% (19/50), which was superior to 20.0% (10/50) in the acupuncture group. CONCLUSION: The comprehensive treatment of fire needles at bones combined with cupping and Tuina, considered as a better therapy for knee osteoarthritis, could improve joint pain, swelling and action function, which is superior to the conventional acupuncture.

[Impacts on biomechanics of senile osteoporosis of kidney deficiency pattern treated with acupuncture and Tuina therapy].

OBJECTIVE: To explore the mechanism on senile osteoporosis of kidney deficiency pattern in the prevention and treatment with acupuncture and Tuina therapy. METHODS: Sixty-four cases were randomized into an acupuncture and Tuina therapy group (group A, 34 cases) and a calcium carbonate group (group B, 30 cases). In group A, the patients were treated with acupuncture at the distal acupoints and Tuina therapy in the local area. Acupuncture was applied to bilateral Weizhong (BL 40) and Taixi (KI 3). Tuina therapy was given in the lumbar region. Totally 30 treatments were required. In group B, the Calcium Carbonate D3 were prescribed for oral administration, continuously for 12 weeks. The changes in lumbar curvature, lumbar lordosis index, sacral inclination angle, lumbosacral angle, L5 vertebral index and L3 vertebral index of lumbar biomechanical indices were observed and compared before and after treatment in two groups. RESULTS: In group A, the lumbar lordosis index and sacral inclination angle were increased after treatment, indicating the statistical significant differences before and after treatment (P < 0.05, P < 0.01). The improvement of lumbar lordosis index in group A was superior to that in group B [(19.59 +/- 19.16)mm vs (14.47 +/- 13.28)mm, P < 0.05]. After treatment, in group B, L5 vertebral index was improved as compared with that before treatment (P < 0.05). It was required to have a study on the reasons of its lumbar morphological change. CONCLUSION: The acupuncture and Tuina therapy regulate the lumbar biomechanical structure through the positive stress stimulation and reconstruct the mechanical equilibrium of the lumbar vertebra. It plays the active significance in the prevention and treatment of osteoporosis.

[Double-center randomized controlled trial on post-stroke shoulder pain treated by electroacupuncture combined with Tuina].

OBJECTIVE: To evaluate clinical therapeutic effect of post-stroke shoulder pain treated by acupuncture combined with Tuina. METHODS: Three hundred cases of post-stroke shoulder pain were randomly divided into an acupuncture and Tuina group and a rehabilitation group by double-center randomized controlled clinical trial method. In acupuncture and Tuina group, normalized electroacupuncture and Tuina therapy were applied, that was electroacupuncture at main points, such as Chize (LU 5), Quze (PC 3), Shaohai (HT 3), Jianyu (LI 15), Jianliao (TE 14) and Jianjing (GB 21),etc., combined with traditional Tuina manipulations; in rehabilitation group, the rehabilitation methods such as the electrostimulation through nervus cutaneus and the squeezing and stabilizing manipulations of Proprioceptive Neuromuscular Facilitation (PNF), etc. were applied. The treatment courses of both groups were 6 weeks. The main therapeutic effect indices were the Assessment Face Scale (AFS) for pain when shoulder was in passive motion and the Fugl-Meyer Motor Assessment for upper limbs active function; the secondary indices were the moditied Rankin Scale (mRS) and the clinical incidences of shoulder-hand syndrome of hemiplegia and shoulder joint subluxation of hemiplegia. RESULTS: After 6 weeks treatment and 12 weeks follow-up, AFS score, Fugl-Meyer motor assessment of upper limbs active function and mRS evaluation in acupuncture and Tuina group were more obviously improved than those in rehabilitation group (P < 0.05, P < 0.01). Although the clinical incidences of shoulder-hand syndrome of hemiplegia and shoulder joint subluxation of hemiplegia in acupuncture and Tuina group was equal to those in rehabilitation group [3.55% (5/141) vs 8.45% (12/142), 1.42% (2/141) vs 5.63% (8/142), both P > 0.05], the data indicated that there was a superiority tendency in acupuncture and Tuina group. CONCLUSION: The combined therapy of electroacupuncture and Tuina is a normative manipulation, and the therapeutic effect is satisfying for post-stroke shoulder pain, superior to that of comprehensive rehabilitation treatment.

[Effect of Tuina at breast on postpartum lactation].

OBJECTIVE: To explore the efficacy of Tuina for postpartum lactation and work out a optimal protocol involved. METHODS: With a randomized, controlled and clinical method, 84 primiparas were divided into a Tuina group and a control group. While patients in the control group received rooming-in conventional managements, those in the Tuina group were additionally treated with Tuina, including local manipulations on breasts combined with acupoint manipulations. The colostrum-time, lactation quantity and prolactin were observed to make the comparisons between two groups. RESULTS: The scores of lactation quantity after 1th, 2nd, 3rd of the treatment were 1.660 +/- 0.785, 2.530 +/- 1.030, 2.880 +/- 1.171 in Tuina group and 1.270 +/- 0.533, 1.460 +/- 0.811, 1.500 +/- 0.583 in control group respectively, where there were significant differences in each time stage between two groups (all P < 0.001). The time of colostrum was (21.6 6 +/- 10.508) h in the Tuina group and (22.5 +/- 9.762) h in the control group, in which the difference was not statistically significant (P > 0.05). The levels of prolactin (314.35 +/- 110.37) ng/mL and (321.56 +/- 109.61) ng/mL in Tuina group, (385.78 +/- 85.19) ng/mL and (340.12 +/- 103.10) ng/mL in control group before and after treatment, there were no significant differences (both P > 0.05). CONCLUSION: Postpartum Tuina on breasts could increase the quantity of lactation and delay the decreasing of the levels of prolactin, which contributes primiparas to lactate more and sooner.

High frequency spectral components after secobarbital: the contribution of muscular origin--a study with MEG/EEG.

OBJECTIVES: Previously we found that benzodiazepines not only provoke beta-activity in the EEG, but also higher frequency activity. Knowing the origin of this high frequency activity is crucial if localisation of epileptogenic brain tissue is the query. We attempt to differentiate cerebral from muscular origin of such activity. METHODS: We postulate that EEG and MEG have similar sensitivity to brain activity, but different sensitivity to muscle activity, and compare co-recorded EEG and MEG signals in a group of five patients who had received short-lasting barbiturates to induce sleep. We performed principal components analysis over time and subtract the results for MEG from the EEG to see where the frequency spectra differ. RESULTS: The EEG showed activity in the gamma bands up to 270Hz for all patients; the MEG significantly less. We find no differences in the lower frequency bands. Topographically the differences localized over the frontotemporal regions. CONCLUSIONS: In the EEG benzodiazepines and/or barbiturates are not only associated with frequencies in the beta band, but also with wide range gamma activity. The latter seems to be of muscular origin. SIGNIFICANCE: Our study suggests that gamma activity in such measurements may not be cerebral in origin. MEG is less susceptible to contamination from muscle activity than the EEG.

[Observation on influence of bone metabolism biochemical indices of senile osteoporosis treated with distant acupuncture and nearby tuina].

OBJECTIVE: To observe the influence of bone metabolism biochemical indices of senile osteoporosis treated with distant acupuncture and nearby tuina, and to probe the better therapy for senile osteoporosis. METHODS: Sixty-four cases were randomly divided into an observation group and a control group. In control group (30 cases), Calcium Carbonate D, Tablet was orally taken for 12 weeks continuously. In observation group (34 cases), "distant acupuncture and nearby tuina" therapy were applied, that was puncture at Weizhong (BL 40) and Taixi (KI 3), and tuina therapy on lumber region, 30 times treatment was applied totally. The changes of blood calcium (Ca), phosphorus (P), ALP, BGP and urine DPD in bone metabolism biochemical indices were observed and compared before and after treatment in two groups. RESULTS: There were significant differences in P, BGP, DPD in bone metabolism biochemical indices in both groups before and after treatment (P < 0.05, P < 0.01), the contents of blood P decreased, blood BGP increased and urine DPD decreased. But, there were no significant differences in above indices between two groups. CONCLUSION: The calcium with oral administration and "distant acupuncture and nearby tuina" therapy can effectively improve the bone metabolism biochemical indices of senile osteoporosis, and they have positive significance in prevention of osteoporosis, however, it hasn't proved that which method is better for calcium protecting.

Barbiturate detection in oral fluid, plasma, and urine.

BACKGROUND: Although current abuse of barbiturates is low compared with other classes of abused drugs, their narrow margin of safety, risk of dependence, and abuse liability remain a health concern. Limited information is available on the disposition of barbiturates in different biologic matrices. OBJECTIVE: The authors conducted a clinical study of the disposition of barbiturates in oral fluid, plasma, and urine after single-dose administration to healthy subjects. METHODS: Three parallel groups of 15 subjects were administered a single oral dose of one barbiturate: butalbital (50 mg), Phenobarbital (30 mg), or sodium secobarbital (100 mg). Subjects remained at the clinic for two confinement periods; the first was -1 to 36 hours postdose and again at 48 to 52 hours. Oral fluid specimens were collected by bilateral collection (Intercept; one on each side of the mouth simultaneously). Blood specimens were obtained by venipuncture and urine specimens were collected through separate collection pools of varying periods. Oral fluid specimens were analyzed for barbiturates by liquid chromatography-tandem mass spectroscopy with a limit of quantitation of 8 ng/mL. Plasma and urine specimens were analyzed by gas chromatography-mass spectroscopy with a limit of quantitation of 100 ng/mL. RESULTS: Barbiturate side effects included dizziness, drowsiness, and somnolence. All effects resolved spontaneously without medical intervention. The three barbiturates were detectable in oral fluid and plasma within 15 to 60 minutes of administration and in the first urine pooled collection at 2 hours. Butalbital and Phenobarbital remained detectable in all specimens through 48 to 52 hours, whereas secobarbital was frequently negative in the last collection. Oral fluid to plasma ratios appeared stable over the 1- to 48-hour collection period. CONCLUSION: This study demonstrated that single, oral therapeutic doses of butalbital, Phenobarbital, and secobarbital were excreted in readily detectable concentrations in oral fluid over a period of approximately 2 days. Oral fluid patterns of appearance and elimination were similar to that observed for plasma and urine.

Postdischarge secobarbital after ED migraine treatment decreases pain and improves resolution.

OBJECTIVE: The objective of the study was to determine whether the addition of postdischarge oral secobarbital to standard emergency department (ED) migraine headache therapy improves pain relief and headache resolution compared with placebo. SETTING: The setting is an urban ED with 70 000 yearly visits. METHODS: This is an Institutional Review Board-approved, randomized, nonconsecutive, double-blinded, concealed, and placebo-controlled clinical trial. Patients with a clinical diagnosis of migraine underwent standard ED treatment and were discharged with 2 tablets of either secobarbital 100 mg or placebo. At home arrival, subjects recorded headache pain on a visual analog scale (VAS), took 1 tablet, and went to bed, taking the second tablet after 1 hour if not asleep. Upon awakening, subjects completed a second VAS and survey. STATISTICAL ANALYSIS: The VAS data were analyzed using 2-tailed t test with unequal variance. Headache resolution data were analyzed using Fisher exact test. RESULTS: Fifty subjects were enrolled. Complete data and follow-up were available for 30 subjects (60%). Fourteen subjects received placebo; 16 received secobarbital. Secobarbital subjects reported an average headache pain decrease of 25 mm (-13 to -38) compared with an average increase of 3 mm (-13 to 19) in the placebo group (P = .01). Ninety-four percent of the secobarbital group vs 50% of the placebo group had complete or partial headache resolution (P < .02). All subjects in the secobarbital group reported some relief. CONCLUSIONS: Addition of postdischarge oral secobarbital to a standard ED migraine treatment regimen decreased headache pain at 24 hours after discharge and improved the rate of headache resolution compared with placebo.

Wada test using secobarbital sodium (Ional) to determine language dominance.

The intracarotid sodium amobarbital (Amytal) test, the Wada test, remains an efficient test for evaluation of language and memory function. However, due to a world shortage of amobarbital, it has become necessary to investigate the use of alternatives. We report the efficacy of the Wada test using secobarbital sodium (Ional) in determining language dominance. An accurate determination of language dominance was required in 43 patients preoperatively at our institution. Patients underwent the Wada test using secobarbital sodium, effectiveness and safety were assessed. Patients were monitored for vital signs (blood pressure, respiratory rates, heart rates and saturation of oxygen). Ten patients were further monitored for continuous intra-arterial blood pressure and monitored with scalp electroencephalography (EEG). Language dominance was determined by the Wada test with secobarbital sodium in all patients. Total volume of secobarbital sodium injected was 10-25 mg (mean 16.5 ± 3.2 mg). Changes in vital signs were minimal and any induced neurological deficits completely disappeared within 8 min. On EEG records, induced theta waves immediately appeared on the ipsilateral side of the intra-arterial injection and disappeared within 6 min. One patient described a scintillating scotoma (sensation of shimmering light in his eyes) at the moment of injection; another experienced an epileptic episode during the test and recovered after 6 min. No adverse events were observed in the remaining 41 cases. We propose secobarbital sodium as a safe and reliable alternative to sodium amobarbital used in the Wada test to determine language dominance.

Quantitation of amobarbital, butalbital, pentobarbital, phenobarbital, and secobarbital in urine, serum, and plasma using gas chromatography-mass spectrometry (GC-MS).

Barbiturates are central nervous system depressants with sedative and hypnotic properties. Some barbiturates, with longer half-lives, are used as anticonvulsants. Their mechanism of action includes activation of gamma-aminobutyric acid (GABA) mediated neuronal transmission inhibition. Clinically used barbiturates include amobarbital, butalbital, pentobarbital, phenobarbital, secobarbital, and thiopental. Besides their therapeutic use, barbiturates are commonly abused. Their analysis is useful for both clinical and forensic proposes. Gas chromatography mass spectrometry is a commonly used method for the analysis of barbiturates. In the method described here, barbiturates from serum, plasma, or urine are extracted using an acidic phosphate buffer and methylene chloride. Barbital is used as an internal standard. The organic extract is dried and reconstituted with mixture of trimethylanilinium hydroxide (TMAH) and ethylacetate. The extract is injected into a gas chromatogram mass spectrometer where it undergoes "flash methylation" in the hot injection port. Selective ion monitoring and relative retention times are used for the identification and quantitation of barbiturates.

Interpretations of the TDxFLx calibration data of the abused drugs.

The useful TDxFLx calibration data was obtained for the interpretation of the interactions of the abused drugs to sheep antiserum protein. The antibody of TDxFLx calibrators was prepared from sheep antiserum. Furthermore these data can be used to interpret the abused drug-protein binding phenomena in human body and the TDxFLx screening results of the abused drugs in urine samples. TDxFLx system uses fluorescence polarization immunoassay technique that is a competitive binding immunoassay methodology to allow tracer-labeled antigen (*Drug) and patient antigen (Drug) to compete for the same binding sites on the antibody molecules of sheep antiserum. To obtain the binding parameters, binding constant (K) and number of independent binding site (n), generally, Scatchard equation is used. This Scatchard equation is expressed in the concentration terms of free drug, bound drug, and protein (antibody). The binding parameters can not be obtained by applying the TDxFLx calibration data to the Scatchard equation directly because the TDxFLx calibration data are composed of the fluorescence polarization and the total drug concentrations. To obtain the binding parameters from the TDxFLx calibration data the new useful equation which was expressed in the total concentrations of drug and fluorescence polarization should be derived. Derivation of new equation was based on the Scatchard equation. The TDxFLx calibration data was curve fitted to the derived equation using KaleidaGraph program and Macintosh computer. The binding constant (K) and the number (n(P(t))) of binding site of 11-nor-delta(9)-tetrahydrocannabinol-9-carboxylic acid (COOH.THC) on the antibody were 1.14 x 10(8)l/mole and 4.04 x 10(-7)M, respectively. The binding constant and the number (n(P(t))) of binding site of amphetamine were 5.15 x 10(5)l/mole and 2.05 x 10(6)M, respectively. In case of COOH.THC the fluorescence polarization decreased linearly with the concentration. However, in case of amphetamine or the other three abused drugs the fluorescence polarizations decreased exponentially with their concentrations.

Barbiturates induce mitochondrial depolarization and potentiate excitotoxic neuronal death.

Barbiturates are widely used as anesthetics, anticonvulsants, and neuroprotective agents. However, barbiturates may also inhibit mitochondrial respiration, and mitochondrial inhibitors are known to potentiate NMDA receptor-mediated neurotoxicity. Here we used rat cortical cultures to examine the effect of barbiturates on neuronal mitochondria and responses to NMDA receptor stimulation. The barbiturates tested, secobarbital, amobarbital, and thiamylal, each potentiated NMDA-induced neuron death at barbiturate concentrations relevant to clinical and experimental use (100-300 microm). By using rhodamine-123 under quenching conditions, barbiturates in this concentration range were shown to depolarize neuronal mitochondria and greatly amplify NMDA-induced mitochondrial depolarization. Barbiturate-induced mitochondrial depolarization was increased by the ATP synthase inhibitor oligomycin, indicating that barbiturates act by inhibiting electron transport sufficiently to cause ATP synthase reversal. Barbiturates similarly amplified the effects of NMDA on cytoplasmic free calcium concentrations. The cell-impermeant barbiturate N-glucoside amobarbital did not influence mitochondrial potential or potentiate NMDA neurotoxicity or calcium responses. However, all of the barbiturates attenuated NMDA-induced calcium elevations and cell death when present at millimolar concentrations. Whole-cell patch-clamp studies showed that these effects may be attributable to actions at the cell membrane, resulting in a block of NMDA-induced current flux at millimolar barbiturate concentrations. Together, these findings reconcile previous reports of opposing effects on barbiturates on NMDA neurotoxicity and show that barbiturate effects on neuronal mitochondria can be functionally significant. Effects of barbiturates on neuronal mitochondria should be considered in experimental and clinical application of these drugs.

Isotopic analogs as internal standards for quantitative analyses by GC/MS--evaluation of cross-contribution to ions designated for the analyte and the isotopic internal standard.

Isotopic analogs of the analytes are currently preferred internal standards (IS) for quantitative analyses of drugs and their metabolites in biological matrices by GC/MS procedures. Contributions of the analyte and the IS to the intensities of ions designated for the IS and the analyte, respectively--an undesirable phenomenon termed "cross-contribution"--greatly weakens the effectiveness of this approach. The cross-contribution phenomenon has been, in the past, evaluated by a "direct measurement" approach, in which intensities of interested ions were measured in two separate experiments using equal quantities of the analyte and the IS. Alternate procedures that may generate improved results are hereby studied. For the "improved direct measurement" approach, ion intensity data derived from the previously reported direct measurement procedure are first normalized before being used to calculate the extent of cross-contribution. An "internal standard" approach is also developed, in which a set amount of a third compound is incorporated into these two separate experiments, thus allowing corrections of ion intensity data that are imbedded with variations inherent to separate experiments. Finally, a "standard addition" approach, involving a series "addition" of "standards", generates multiple data points; thus, providing a mechanism to validate the resulting cross-contribution data. Secobarbital/(2)H(5)-secobarbital and secobarbital/(13)C(4)-secobarbital pairs are adapted as the exemplar systems for this study.

Isotopic analogues as internal standards for quantitative analyses of drugs and metabolites by GC-MS--nonlinear calibration approaches.

In order to achieve accurate quantitation of drugs and metabolites (analytes) in complex matrices, 2H- (and less commonly 13C-) labeled analogues of the analytes are now routinely adapted as the internal standards (IS) using linear calibration models to fit data generated by selected ion monitoring gas chromatography-mass spectrometry (GC-MS) protocols. In this study, the effects of cross-contribution (contribution of the IS to the intensity of the ion designated for the analyte and vice versa) on the linearity of the calibration data are examined. Nonlinear approaches that may address this problem are also studied. Two ion pairs (one with least and one with significant cross-contribution) from each of the following analyte/IS pairs are used as the exemplar systems for this study: butalbital/13C4-butalbital, butalbital/2H5-butalbital, secobarbital/13C4-secobarbital, and secobarbital/2H5-secobarbital. Analyte/IS ion intensity ratios of a series of standard solutions are correlated with the analyte/IS concentration ratios using one-point, multiple-point (unweighted and weighted) linear, and hyperbolic functions. The one-point calibration approach produces excellent calibration results in treating data derived from ion pairs with no significant cross contribution. In cases where significant cross-contribution exists, results derived from the one-point approach show, as expected, significant deviations at both ends of the concentration range. With the cross-contribution phenomenon accounted for, the hyperbolic calibration model is clearly more effective in fitting calibration data at both the lower and higher analyte concentration ends, thus significantly lowering the detection limit and extending the calibration range to a higher level. However, the calibration range cannot be extended indefinitely. At the low concentration end, noise-to-signal ratio and the cross-contribution of the IS to the intensity of the ion designated for the analyte, however insignificant, will incrementally reduce the quality of the observed ion intensity and intensity ratio data. At the high concentration end, detection saturation and the cross-contribution of the analyte to the intensity of the ion designated for the IS, however insignificant, will incrementally decrease the "slope" of the calibration curve. Thus, acceptable sensitivity (increase in analyte/IS ion-pair intensity ratio per unit increase in analyte concentration) of the calibration curve will become the limiting factor.