Longitudinal Analysis of the Effect of Inflammation on Voriconazole Trough Concentrations.

Voriconazole (VCZ) exhibits great inter- and intrapatient variability. The latter variation cannot exclusively be explained by concomitant medications, liver disease or dysfunction, and genetic polymorphisms in cytochrome P450 2C19 (CYP2C19). We hypothesized that inflammatory response in patients under VCZ medication might also influence this fluctuation in concentrations. In this study, we explored the association between inflammation, reflected by the C-reactive protein (CRP) concentration, and VCZ trough concentrations over time. A retrospective analysis of data was performed for patients with more than one steady-state VCZ trough concentration and a CRP concentration measured on the same day. A longitudinal analysis was used for series of observations obtained from many study participants over time. The approach involved inclusion of random effects and autocorrelation in linear models to reflect within-person cross-time correlation. A total of 50 patients were eligible for the study, resulting in 139 observations (paired VCZ and CRP concentrations) for the analysis, ranging from 2 to 6 observations per study participant. Inflammation, marked by the CRP concentration, had a significant association with VCZ trough concentrations (P < 0.001). Covariates such as age and interacting comedication ([es]omeprazole), also showed a significant correlation between VCZ and CRP concentrations (P < 0.05). The intrapatient variation of trough concentrations of VCZ was 1.401 (confidence interval [CI], 0.881 to 2.567), and the interpatient variation was 1.756 (CI, 0.934 to 4.440). The autocorrelation between VCZ trough concentrations at two sequential time points was calculated at 0.71 (CI, 0.51 to 0.92). The inflammatory response appears to play a significant role in the largely unpredictable pharmacokinetics of VCZ, especially in patients with high inflammatory response, as reflected by high CRP concentrations.

Long-term exposure to circulating platinum is associated with late effects of treatment in testicular cancer survivors.

BACKGROUND: The success of cisplatin-based (Platinol, Bristol-Myers Squibb Company, New York, NY, USA) chemotherapy for testicular cancer comes at the price of long-term and late effects related to healthy tissue damage. We assessed and modelled serum platinum (Pt) decay after chemotherapy and determined relationships between long-term circulating Pt levels and known late effects. PATIENTS AND METHODS: In 99 testicular cancer survivors, treated with cisplatin-based chemotherapy, serum and 24-h urine samples were collected during follow-up (1-13 years after treatment). To build a population pharmacokinetic model, measured Pt data were simultaneously analysed, together with cisplatin dose, age, weight and height using the NONMEM software. Based on this model, area under the curve between 1 and 3 years after treatment (Pt AUC1-3 years) was calculated for each patient. Predicted long-term Pt exposure was related to renal function and to late effects of treatment assessed median 9 (3-15) years after chemotherapy. RESULTS: Decay of Pt was best described by a two-compartment model. Mean terminal T1/2 was 3.7 (range 2.5-5.2) years. Pt AUC1-3 years correlated with cumulative cisplatin dose, and creatinine clearance before and 1 year after treatment. Patients with paraesthesia had higher Pt AUC1-3 years (30.9 versus 27.0 µg/l month) compared with those without paraesthesia (P = 0.021). Patients with hypogonadism, elevated LDL-cholesterol levels or hypertension also had higher Pt AUC1-3 years. CONCLUSIONS: Renal function before and after cisplatin treatment is an important determinant of long-term Pt exposure. Known long-term effects of testicular cancer treatment, such as paraesthesia, hypogonadism, hypercholesterolaemia and hypertension, are associated with long-term circulating Pt exposure.

Five year results of an international proficiency testing programme for measurement of antifungal drug concentrations.

OBJECTIVES: Since 2007 the Dutch Association for Quality Assessment in Therapeutic Drug Monitoring (KKGT) has organized an international interlaboratory proficiency testing (PT) programme for measurement of antifungal drugs in plasma. We describe the 5 year results of the laboratories' performance. METHODS: Twice a year, laboratories received a set of blind plasma samples containing low or high concentrations of fluconazole, itraconazole, hydroxyitraconazole, posaconazole, voriconazole and flucytosine. Participating laboratories were asked to report their results within 6 weeks after dispatch and provide details of their analytical methods. Results deviating >20% from the weighed-in concentration were considered inaccurate. Four-way ANOVA was performed to assess the effect of antifungal drug measured, concentration, analytical method and performing laboratory on the absolute inaccuracy. In 2012, a questionnaire based on the CLSI guidelines was dispatched with the request to provide input on sources of error. RESULTS: Fifty-seven laboratories (13 countries) reported 2251 results (287 fluconazole, 451 itraconazole, 348 hydroxyitraconazole, 402 posaconazole, 652 voriconazole and 111 flucytosine) in 5 years. Analyses were performed using HPLC (55.0%), LC-MS(/MS) (43.4%), UPLC (1.4%) or GC-MS (0.2%). Overall, 432 (19.2%) analyses were inaccurate. The performing laboratory was the only factor clearly associated with inaccuracies. The questionnaire results indicated that laboratories encounter significant problems analysing low concentrations (15.4% of all inaccuracies). CONCLUSIONS: Results of the PT programme suggest that one out of five measurements is inaccurate. The performing laboratory is the main determinant of inaccuracy, suggesting that internal quality assurance is pivotal in preventing inaccuracies, irrespective of the antifungal drug measured, concentration and analytical equipment.

Simultaneous determination of rifampicin, clarithromycin and their metabolites in dried blood spots using LC-MS/MS.

INTRODUCTION: Rifampicin (RIF) and clarithromycin (CLR) are common drugs for the treatment of infections like Mycobacterium tuberculosis and Mycobacterium ulcerans. Treatment for these diseases are long-term and the individual pharmacokinetic variation, drug-drug interactions or non-adherence may introduce sub-therapeutic exposure or toxicity. The application of therapeutic drug monitoring (TDM) can be used to ensure efficacy and avoid toxicity. With the use of dried blood spot (DBS), TDM may be feasible in rural areas. During DBS method development, unexpected interactions or matrix effects may be encountered due to endogenous components in the blood. Another complication compared to plasma analysis is that RIF can form chelate complexes with ferric ions or can bind with hemes, which are potentially present in the extracts of dried blood spots. METHODS: The investigation focused on the interaction between RIF and the endogenous components of the DBS. The use of ethylenediaminetetraacetic acid (EDTA) and deferoxamine (DFX) as chelator agents to improve recoveries and matrix effects were investigated. A rapid analytical method was developed and validated to quantify RIF and CLR and their active metabolites desacetyl rifampicin (DAc-RIF) and 14-hydroxyclarythromcin (14OH-CLR) in DBS samples. A clinical application study was performed in tuberculosis patients by comparing DBS concentrations with plasma concentrations. RESULTS: The interaction between RIF and the DBS matrix was avoided using the complexing agents EDTA and DFX, which improved recoveries and matrix effects. The developed sample procedure resulted in a simple and fast method for the simultaneous quantification of RIF, CLR and their metabolites in DBS samples. High stability was observed as all four substances were stable at ambient temperature for 2 months. Deming regression analysis of the clinical application study showed no significant differences for RIF, DAc-RIF, CLR and 14OH-CLR between patient plasma and DBS analysis. The slopes of the correlation lines between DBS and plasma concentrations of RIF, DAc-RIF, CLR and 14OH-CLR were 0.90, 0.99, 0.80 and 1.09 respectively. High correlations between plasma and DBS concentrations were observed for RIF (R(2)=0.9076), CLR (R(2)=0.9752) and 14OH-CLR (R(2)=0.9421). Lower correlation was found for DAc-RIF (R(2) of 0.6856). CONCLUSION: The validated method is applicable for TDM of RIF, CLR and their active metabolites. The stability of the DBS at high temperatures can facilitate the TDM and pharmacokinetic studies of RIF and CLR even in resource limited areas. The role of EDTA and DFX as complexing agents in the extraction was well investigated and may provide a solution for potential applications to other DBS analytical methods.

Pharmacokinetic variability of clarithromycin and differences in CYP3A4 activity in patients with cystic fibrosis.

BACKGROUND: To investigate the correlation between CYP3A4/5 activity and clarithromycin metabolism, and between CYP3A activity and CYP3A genotype. METHODS: This is an open-label, prospective pharmacokinetic study evaluating CYP3A activity using The Erythromycin Breath Test. Eight blood samples were collected within 12h after clarithromycin 500 mg was administered orally. The clarithromycin concentrations were measured by liquid chromatography-tandem mass spectrometry. AUC, Tmax and Cmax were calculated. Selected Single Nucleotide polymorphisms in CYP3A4/5 genes were assessed by PCR and single base extension. RESULTS: Twenty-one chronically infected patients were included. An 8-fold variation in the CYP3A4 activity, 10-fold variation in AUC for clarithromycin (median 881 µg/mL × min), and a 16-fold variation in Cmax for clarithromycin (median 3.4 µg/mL) were found. A linear correlation between the CYP3A4-activity and clarithromycin metabolism was demonstrated (P < 0.05). CONCLUSION: The large variation in the clarithromycin pharmacokinetics in cystic fibrosis patients may cause treatment failure. The Erythromycin Breath Test could be valuable in identifying cystic fibrosis patients in risk of treatment failure/drug toxicity.

Clinical validation of the analysis of linezolid and clarithromycin in oral fluid of patients with multidrug-resistant tuberculosis.

Linezolid plays an increasingly important role in the treatment of multidrug-resistant tuberculosis (MDR-TB). However, patients should be carefully monitored due to time- and dose-dependent toxicity. Clarithromycin plays a more modest role. Therapeutic drug monitoring may contribute to assessment of treatment regimens, helping to reduce toxicity while maintaining adequate drug exposure. Oral fluid sampling could provide a welcome alternative in cases where conventional plasma sampling is not possible or desirable. The aim of this study was to clinically validate the analysis of linezolid and clarithromycin and its metabolite hydroxyclarithromycin in oral fluid of patients with multidrug-resistant tuberculosis. Serum and oral fluid samples were simultaneously obtained and analyzed by using validated methods, after extensive cross-validation between the two matrices. Passing-Bablok regressions and Bland-Altman analysis showed that oral fluid analysis of linezolid and clarithromycin appeared to be suitable for therapeutic drug monitoring in MDR-TB patients. No correction factor is needed for the interpretation of linezolid oral fluid concentrations with a ratio of the linezolid concentration in serum to that in oral fluid of 0.97 (95% confidence interval [CI], 0.92 to 1.02). However, the clarithromycin concentration serum/clarithromycin concentration in oral fluid ratio is 3.07 (95% CI, 2.45 to 3.69). Analysis of hydroxyclarithromycin in oral fluid was not possible in this study due to a nonlinear relationship between the concentration in serum and that in oral fluid. In conclusion, the analysis of linezolid (no correction factor) and clarithromycin (correction factor of 3) in oral fluid is applicable for therapeutic drug monitoring in cases of multidrug-resistant tuberculosis as an alternative to conventional serum sampling. Easy sampling using a noninvasive technique may facilitate therapeutic drug monitoring for specific patient categories.

Troubleshooting carry-over of LC-MS/MS method for rifampicin, clarithromycin and metabolites in human plasma.

Clarithromycin and rifampicin are used for the treatment of Mycobacteria. Pharmacokinetic drug interaction is possibly due to the influence of the two drugs on the liver enzymes. Using a Hypurity Aquastar C18 column (50mm×2.1mm×5µm) for liquid chromatography including a polar end-capped phase for the determination of clarithromycin, rifampicin and their metabolites together in plasma using LC-MS/MS resulted in a substantial carry-over. As a consequence, the throughput of the method is not assured. Using a step-by-step troubleshooting procedure, such carry-over was found originating from column memory effect. With the use of another type of C18 column, the carry-over is eliminated. Due to the absence of carry-over, the analytical concentration ranges are extended and are therefore more appropriate for the analysis of patient samples. The method was re-validated for linearity, reproducibility and dilution integrity.

Dried blood spot analysis for therapeutic drug monitoring of linezolid in patients with multidrug-resistant tuberculosis.

Linezolid is a promising antimicrobial agent for the treatment of multidrug-resistant tuberculosis (MDR-TB), but its use is limited by toxicity. Therapeutic drug monitoring (TDM) may help to minimize toxicity while adequate drug exposure is maintained. Conventional plasma sampling and monitoring might be hindered in many parts of the world by logistical problems that may be solved by dried blood spot (DBS) sampling. The aim of this study was to develop and validate a novel method for TDM of linezolid in MDR-TB patients using DBS sampling. Plasma, venous DBS, and capillary DBS specimens were obtained simultaneously from eight patients receiving linezolid. A DBS sampling method was developed and clinically validated by comparing DBS with plasma results using Passing-Bablok regression and Bland-Altman analysis. This study showed that DBS analysis was reproducible and robust. Accuracy and between- and within-day precision values from three validations presented as bias and coefficient of variation (CV) were less than 17.2% for the lower limit of quantification and less than 7.8% for other levels. The method showed a high recovery of approximately 95% and a low matrix effect of less than 8.7%. DBS specimens were stable at 37°C for 2 months and at 50°C for 1 week. The ratio of the concentration of linezolid in DBS samples to that in plasma was 1.2 (95% confidence interval [CI], 1.12 to 1.27). Linezolid exposure calculated from concentrations DBS samples and plasma showed good agreement. In conclusion, DBS analysis of linezolid is a promising tool to optimize linezolid treatment in MDR-TB patients. An easy sampling procedure and high sample stability may facilitate TDM, even in underdeveloped countries with limited resources and where conventional plasma sampling is not feasible.

Dried blood spots: a new tool for tuberculosis treatment optimization.

Tuberculosis (TB) is a high-burden infectious disease, especially in low and middle-income countries. The efforts to eliminate this disease are challenged by the emergence of multidrug resistance and TB-HIV coinfection. The cumulative knowledge on pharmacokinetics/ pharmacodynamics of antituberculosis agents has recently encouraged therapeutic drug monitoring (TDM) in patient care. However, logistical problems related to conventional sampling limit the application of TDM in research-oriented institutions. Dried blood spot (DBS) compared with conventional venous blood sampling has the advantages of easier sampling, storage and transportation, thus enabling the application of TDM even in remote areas. In addition, DBS with its lower biohazardous risk can be safely performed in a high HIV prevalence area, which also tends to have a high TB burden. Another benefit of DBS sampling is that it requires a smaller blood volume than conventional sampling and is highly recommended for application in pediatric TB. A limitation of DBS is that additional considerations are required for analysis method development and validation. The accuracy of the DBS method is influenced by a number of factors that need to be thoroughly examined in method development and validation. Further, the agreement between DBS and plasma/serum concentrations is not always understood and further investigations are required.

Determination of moxifloxacin in dried blood spots using LC-MS/MS and the impact of the hematocrit and blood volume.

Moxifloxacin (MFX) is a potential oral agent use in the treatment of multidrug-resistance tuberculosis (MDR-TB). Due to variability in pharmacokinetics and in vitro susceptibility of causative bacteria, therapeutic drug monitoring (TDM) of MFX is recommended. Conventional plasma sampling for TDM is facing logistical challenges, especially in limited resource areas, and dried blood spots (DBS) sampling may offer a chance to overcome this problem. The objective of this study was to develop a LC-MS/MS method for determination of MFX in dried blood spots (DBS) that is applicable for TDM. The influence of paper type, the hematocrit (Hct) and the blood volume per spot (V(b)) on the estimated blood volume in a disc (V(est)) was investigated. The extracts of 8mm diameter discs punched out from DBS were analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS) with cyanoimipramin as internal standard. The method was validated with respect to selectivity, linearity, accuracy, precision, sensitivity, recovery and stability. The effect of Hct and V(b) on LC-MS/MS analytical result was also investigated. The relationship between MFX concentrations in venous and finger prick DBS and those in plasma was clinically explored. V(est) was highly influenced by Hct while the effect of V(b) appeared to be different among paper types. Calibration curves were linear in the range of 0.05-6.00 mg/L with inter-day and intra-day precisions and biases of less than 11.1%. The recovery was 84.5, 85.1 and 92.6% in response to blood concentration of 0.15, 2.50 and 5.00 mg/L, respectively. A matrix effect of less than 11.9% was observed. MFX in DBS was stable for at least 4 weeks at room condition (temperature of 25°C and humidity of 50%). A large range of Hct value produced a significant analytical bias and it can be corrected with resulting DBS size. A good correlation between DBS and plasma concentrations was observed and comparable results between venous DBS and finger prick DBS was attained. This fully validated method is suitable for determination of MFX in dried blood spot and applicable for TDM.

Evaluation of moxifloxacin for the treatment of tuberculosis: 3 years of experience.

Moxifloxacin (MFX) is a powerful second-line anti-tuberculosis (TB) agent, but the optimal dose has not yet been established and long-term safety data are scarce. We retrospectively reviewed the medical charts of TB patients treated at the Tuberculosis Centre Beatrixoord, University Medical Centre Groningen (Haren, the Netherlands) receiving MFX 400 mg once daily as part of their TB treatment between January 1 2006 and January 1 2009. Safety data and drug-drug interactions were evaluated. Efficacy was predicted based on the area under the concentration-time curve up to 24 h post-dosage (AUC(0-24h))/minimal inhibitory concentration (MIC) ratio. 89 patients were treated with a median dose of 6.9 mg · kg(-1) MFX once daily for a median period of 74 days. Discontinuation of therapy occurred in only three patients due to gastrointestinal side-effects and hypersensitivity. Pharmacokinetic analysis showed an AUC(0-24h)/MIC ratio <100 in eight out of 16 patients. A large variation in protein binding affected the unbound AUC(0-24h) considerably. These data show that MFX treatment was well tolerated in 89 patients receiving a dose of 400 mg once daily for a prolonged period. Considering the variability in (un)bound AUC(0-24h)/MIC ratio, therapeutic drug monitoring is recommended in selected patients (i.e. rifampicin co-medication; MIC ≥ 0.25 mg · L(-1)) to assess optimal therapy.

Pharmacokinetics of rifampin and clarithromycin in patients treated for Mycobacterium ulcerans infection.

In a randomized controlled trial in Ghana, treatment of Mycobacterium ulcerans infection with streptomycin (SM)-rifampin (RIF) for 8 weeks was compared with treatment with SM-RIF for 4 weeks followed by treatment with RIF-clarithromycin (CLA) for 4 weeks. The extent of the interaction of RIF and CLA combined on the pharmacokinetics of the two compounds is unknown in this population and was therefore studied in a subset of patients. Patients received CLA at a dose of 7.5 mg/kg of body weight once daily, rounded to the nearest 125 mg. RIF was administered at a dose of 10 mg/kg, rounded to the nearest 150 mg. SM was given at a dose of 15 mg/kg once daily as an intramuscular injection. Plasma samples were drawn at steady state and analyzed by liquid chromatography-tandem mass spectroscopy. Pharmacokinetic parameters were calculated with the MW/Pharm (version 3.60) program. Comedication with CLA resulted in a 60% statistically nonsignificant increase in the area under the plasma concentration-time curve (AUC) for RIF of 25.8 mg x h/liter (interquartile ratio [IQR], 21.7 to 31.5 mg x h/liter), whereas the AUC of RIF was 15.2 mg x h/liter (IQR, 15.0 to 17.5 mg x h/liter) in patients comedicated with SM (P = 0.09). The median AUCs of CLA and 14-hydroxyclarithromycin (14OH-CLA) were 2.9 mg x h/liter (IQR, 1.5 to 3.8 mg x h/liter) and 8.0 mg x h/liter (IQR, 6.7 to 8.6 mg x h/liter), respectively. The median concentration of CLA was above the MIC of M. ulcerans, but that of 14OH-CLA was not. In further clinical studies, a dose of CLA of 7.5 mg/kg twice daily should be used (or with an extended-release formulation, 15 mg/kg should be used) to ensure higher levels of exposure to CLA and an increase in the time above the MIC compared to those achieved with the currently used dose of 7.5 mg/kg once daily.

Intravenous voriconazole after toxic oral administration.

In a male patient with rhinocerebral invasive aspergillosis, prolonged high-dosage oral administration of voriconazole led to hepatotoxicity combined with a severe cutaneous reaction while intravenous administration in the same patient did not. High concentrations in the portal blood precipitate liver enzyme abnormalities, and therefore, oral administration of voriconazole may have a hepatotoxicity profile different from that of intravenous (i.v.) administration. Intravenously administered voriconazole might still be an option after oral-voriconazole-induced toxicity has resolved.

Method for therapeutic drug monitoring of azole antifungal drugs in human serum using LC/MS/MS.

Fungal infections occur in immunocompromised patients. Azole antifungal agents are used for the prophylaxis and treatment of these infections. The interest in therapeutic drug monitoring azole agents has increased over the last few years. Inter- and intra-patient variability of pharmacokinetics, drug-drug interactions, serum concentration related toxicity and success of therapy has stressed the need of frequently therapeutic drug monitoring of the drugs, belonging to the group of azoles. Therefore a simple, rapid and flexible method of analysis is required. This method is based on the precipitation of proteins in human serum with LC/MS/MS detection. Validation was performed according to the guidelines for bioanalytical method validation of the food and drug administration agency. The four most used azole drugs can be detected in human serum within the clinical relevant serum levels with good accuracy and reproducibility at the limit of quantification. Intra- and inter-day validation demonstrated good accuracy and reproducibility. A rapid, sensitive and flexible LC/MS/MS method has been developed and validated to measure voriconazole (VRZ), fluconazole (FLZ), itraconazole (ITZ) and posaconazole (PSZ) in human serum. This new method is suitable for clinical pharmacokinetic studies and routine monitoring in daily practice.

Neurophysiological evaluation in children with Friedreich's ataxia.

INTRODUCTION: In children with Friedreich's ataxia (FRDA children), clinical ataxia outcomes are hardly substantiated by underlying neurophysiological parameters. In young FRDA children, some reports (based upon International Cooperative Ataxia Rating Scale scores (ICARS)) mention transient neurological improvement upon idebenone treatment. However, these outcomes are obtained with adult instead of pediatric reference values. It is unknown whether age-related neurophysiological parameters can really substantiate neurologic improvement. AIM: In young FRDA children, we aimed to determine longitudinal neurophysiological parameters during idebenone treatment. METHODS: During a two-year study period, 6 genetically proven FRDA children with cardiomyopathy (6-18years) were longitudinally assessed for neurophysiological parameters [sensory evoked potentials (SEPs), F response, peripheral nerve conduction and dynamometry] in association with age-matched ICARS-scores. RESULTS: In all FRDA children, SEPs remained absent during the two-year study period. Peroneal nerve conduction velocity declined (from -1SD to -2SD; p<.05), whereas F responses remained essentially unaltered. Total muscle force and leg muscle force decreased (from -2 to -3SD and -2.5 to -3.5SD; both p<.05) and age-related ICARS-scores deteriorated (median increase +41%; p<.05). CONCLUSION: In FRDA children, age-related neurophysiological and ataxia parameters deteriorate during idebenone treatment. Although we cannot exclude some (subjective) disease stabilization, age-related neurophysiological parameters do not substantiate neurologic improvement.

The relation between the blood benzodiazepine concentration and performance in suspected impaired drivers.

Several experimental studies have shown a negative influence of benzodiazepines on driving skills. The objective of this study is to study the relationship between the blood concentration of benzodiazepines and the influence on performance in field sobriety tests. A retrospective case file evaluation was conducted to select cases of drivers, tested positive for benzodiazepines only in the period from January 1999 to December 2004. Drivers were grouped into the categories sub therapeutic, therapeutic or elevated concentrations. The outcome of the tests (walking, walking after turn, nystagmus, Romberg's test, behavior, pupils and orientation) was binomial. A Chi square test was used to assess differences in proportions of the categorized cases. In total 171 cases were included. Observations of behavior (n=137; p<0.01), walking (n=109; p<0.01), walking after turn (n=89; p=0.02) and Romberg's test (n=88; p<0.05) were significantly related to the benzodiazepine concentration. There was no significant relation between benzodiazepine concentration and effect on pupil size, nystagmus or orientation. The results of our study indicate a relation between the concentration of benzodiazepines and the results of some performance tests. More effort is needed to standardize the tests and to determine the sensitivity and selectivity of the tests for benzodiazepines.

New practical algorithm for modelling analyte recovery in bioanalytical reversed phase and mixed-mode solid phase extraction.

Solid phase extraction (SPE) is a widely used method for sample cleanup and sample concentration in bioanalytical sample preparation. A few methods to model the retention behaviour on SPE cartridges have been described previously but they are either not applicable to ionised species or are not suitable when using multiple wash and elution steps with solvents differing in volume, modifier concentration and acidity. Furthermore, these models were not applied to mixed mode SPE sorbents. In order to overcome these limitations a new SPE modelling algorithm was proposed. The retention behaviour was determined directly on the SPE cartridge by connecting the cartridge online with an HPLC system using a simple but suitable device that was developed and described. The results from these online experiments were used to model the elution behaviour using a quadratic retention function combined with an exponentially modified Gaussian peak shape model to predict analyte recovery under different wash and elution conditions. The validity of the proposed algorithm was tested using practical SPE experiments with an aqueous test mixture as well as with spiked human plasma. Different sequential wash and elution steps were performed using solvents differing in volume and composition. The predicted band shape and recoveries in each collected step were in good agreement with the results obtained from practical experiments. The proposed algorithm is very useful for the description of the SPE behaviour of the analytes on the actual used SPE cartridge and can be used in structural and automated SPE method development.

The relation between the use of psychoactive substances and the severity of the injury in a group of crash-involved drivers admitted to a regional trauma center.

OBJECTIVE: There is much evidence that driving under the influence of alcohol and/or drugs of abuse is related to an increased accident risk. A remaining question is whether the use of psychoactive substances is also related to clinically more severe accidents. The aim of this study is to explore the relationship between the use of psychoactive substances and the injury severity in a group of crash-involved drivers. METHODS: The study group included all injured car drivers, admitted to the regional trauma center, in the period from May 2000 until August 2001. The outcome of interest was the severity of injury, measured by using the Injury Severity Score (ISS). The determinant was the presence of psychoactive substances in blood and urine samples. Psychoactive substances tested for were alcohol, amphetamines, barbiturates, benzodiazepines, cannabis, methadone, opiates, and tricyclic antidepressants in blood and urine. RESULTS: The number of injured car drivers included in this study was 106. Overall, 43% (46/106) of the drivers tested positive for at least one psychoactive substance. Comparison of the means of the log ISS suggests that there is no significant difference between drivers who tested positive for alcohol and/or drugs, compared to drivers tested negative. CONCLUSION: The results of this study support the hypothesis that there is no clear association between use of psychoactive substances and the severity of crash-related injury.

pH adjustment of human blood plasma prior to bioanalytical sample preparation.

pH adjustment in bioanalytical sample preparation concerning ionisable compounds is one of the most common sample treatments. This is often done by mixing an aliquot of the sample with a proper buffer adjusted to the proposed pH. The pH of the resulting mixture however, does not necessarily have to be the same as the pH of the used buffer due to the significant buffer capacity of the sample. Calculation methods from titration technology were adapted and applied to this problem. The acid-base characteristics of human blood plasma and serum samples were determined and used to calculate the pH of buffer-plasma mixtures. Based on these parameters and the characteristics of the used buffers, two alternative methods were described to prepare buffers that lead to the proposed pH when mixed in the right volume ratio with human plasma samples. The resulting pH of several mixtures of different buffers with human blood plasma were in good accordance with the calculated pH. The proposed calculation methods and recommended buffer preparation methods may lead to more robust bioanalytical methods.