Development and validation of a stability indicating RP-HPLC-DAD method for the determination of bromazepam.

A reliable, selective and sensitive stability-indicating RP-HPLC assay was established for the quantitation of bromazepam (BMZ) and one of the degradant and stated potential impurities; 2-(2-amino-5-bromobenzoyl) pyridine (ABP). The assay was accomplished on a C18 column (250 mm × 4.6 mm i.d., 5 µm particle size), and utilizing methanol-water (70: 30, v/v) as the mobile phase, at a flow rate of 1.0 ml min-1. HPLC detection of elute was obtained by a photodiode array detector (DAD) which was set at 230 nm. ICH guidelines were adhered for validation of proposed method regarding specificity, sensitivity, precision, linearity, accuracy, system suitability and robustness. Calibration curves of BMZ and ABP were created in the range of 1-16 µg mL-1 with mean recovery percentage of 100.02 ± 1.245 and 99.74 ± 1.124, and detection limit of 0.20 µg mL-1 and 0.24 µg mL-1 respectively. BMZ stability was inspected under various ICH forced degradation conditions and it was found to be easily degraded in acidic and alkaline conditions. The results revealed the suitability of the described methodology for the quantitation of the impurity (ABP) in a BMZ pure sample. The determination of BMZ in pharmaceutical dosage forms was conducted with the described method and showed mean percentage recovery of 99.39 ± 1.401 and 98.72 ± 1.795 (n = 6), respectively. When comparing the described procedure to a reference HPLC method statistically, no significant differences between the two methods in regard to both accuracy and precision were found.

Stability indicating spectrophotometric methods for quantitative determination of bromazepam and its degradation product.

Four simple, sensitive and selective stability indicating spectrophotometric methods are presented for quantitative determination of the benzodiazepine drug; bromazepam (BMZ) and one of its reported potential impurities and degradation product; 2-(2-amino-5-bromobenzoyl) pyridine (ABP) in methanol. Method A, is isoabsorptive point coupled with D0 method, where good linearity was obtained by measuring the absorbance of BMZ at 264 nm (Aiso) in the concentration range of 2-25 µg mL-1, and the absorbance of ABP at its λmax 396 nm in concentration range of 0.5-24 µg mL-1. Method B, is ratio subtraction; the absorbance was measured at 233 nm for BMZ using 20 µg mL-1 of ABP, while ABP was determined directly at its λmax 396 nm using methanol as a solvent. Method C, was based on measuring the total peak amplitude of the first derivative of the ratio spectra (DD1) of BMZ from 301 to 326 nm using 10 µg mL-1 of ABP as a divisor and determination of ABP at peak amplitude of 293 nm using 5 µg mL-1 of BMZ as a divisor. In method D, ratio difference method, good linearity was achieved for determination of BMZ and ABP by measuring the differences between the amplitudes of ratio spectra at 312 nm and 274 nm and differences between the amplitudes of ratio spectra at 274 nm and 312 nm, respectively. The stability of BMZ was investigated under different ICH recommended forced degradation conditions. The suggested methods were then successfully applied for determination of BMZ in its pharmaceutical formulations.

Analysis of Complexation Interactions between Metal Ions and Drugs under Pseudo-physiological pH Conditions by a High-throughput Screening Method Using a Solid-phase Extraction Cartridge.

A high-throughput screening method for the complexation between metal ions and drugs was established by combining solid-phase extraction (SPE) using a nitrilotriacetic acid (NTA) modified silica spin cartridge with subsequent HPLC analysis. First, a test metal ion solution was passed through the NTA cartridge, then a test drug solution diluted in phosphate buffered saline (pH 7.4) was passed through the metal-chelated NTA cartridge. The complexation behavior between the metal and the drug on the NTA cartridge was evaluated by HPLC quantification of the drug in the SPE eluate. Comprehensive analysis of the complexation behavior between 11 different metal ions and 55 drugs showed that Cu2+, Ni2+, Co2+, Zn2+, Cr3+ and Fe3+ formed complexes with 12, 5, 4, 2, 1 and 1 kinds of drugs, respectively. Bromazepam selectively formed complexes with Cu2+, Ni2+ and Co2+.

Development and Validation of an Analytical Method for the Detection and Quantification of Bromazepam, Clonazepam and Diazepam by UPLC-MS/MS in Surface Water.

The development of analytical methods capable of determining micropollutants is essential for quality control of drinking water. Benzodiazepines, a class of pharmaceuticals with anxiolytic properties, have received increasing attention as micropollutants. The purpose of this study was to develop an analytical method for determination of three benzodiazepine drugs (bromazepam, clonazepam and diazepam) in surface water. For the extraction of the matrix analytes, SPE cartridges (C18, 500 mg/3 mL) were used. The method was validated according to the quality criteria of the USEPA 8000D Validation Guide. The developed and validated method showed recovery values between 57 and 100%, RSD < 20% and R2 > 0.9949. LD ranged between 2.70 and 5.00 ng L-1 for bromazepam and clonazepam respectively whereas LQ was 0.01 µg L-1 for all analytes. The matrix affected the signal intensity of clonazepam thus evidencing the matrix effect by analysis statistic (F test).

Performance Comparison Between Monolithic, Core-Shell, and Totally Porous Particulate Columns for Application in Greener and Faster Chromatography.

Background: The introduction of monolithic rods and core-shell particles as new morphologies of packing materials different from the conventional totally porous particles resulted in a leap forward for performance in LC. Meanwhile, environmental safety has become increasingly important in many areas, especially in industry and research laboratories. Objective: This study compared the efficiencies of commercially available columns of different lengths and diameters when greener chromatographic conditions were utilized. The main purpose of this study is to help practitioners select the most appropriate stationary phase for faster and greener analysis. Methods: The three types of stationary phases were compared in terms of separation efficiency, number of theoretical plates, peak shape, selectivity, resolution, analysis time, mobile phase consideration, and permeability using six drug molecules. Results: Results indicated that core-shell and monolithic stationary phases had superiority over the conventional totally porous particles in terms of efficiency and speed of analysis. Monolithic rods had lower column backpressure and higher permeability, so they are more suitable for higher mobile phase flow rates and viscosities. However, core-shell particles provided enhanced peak shapes and number of theoretical plates. Conclusions: The choice will depend on the main purpose of analysis and the composition of the mobile phase. Compromise must be made to obtain the best trade-off between separation efficiency and analysis speed. Highlights: This study is the first to consider green chromatography concepts for the selection of the best stationary phase of new morphologies.

Forensic toxicological analyses of drugs in tissues in formalin solutions and in fixatives.

Forensic toxicological drug analyses of human specimens are usually performed immediately after autopsy or on frozen preserved tissues. Occasionally, cases require analysis of drugs from tissues fixed in formalin solution. To improve the estimation of the level of drug in tissues following formalin fixation, we studied drug concentrations in human tissues, liver and kidney, that were collected from a drug-positive autopsy case. Parts of tissues were preserved in formalin solution for 1, 3, 6 and 13 months. Tissues obtained before and after preservation, along with tissue-exposed fixatives, were assayed using gas chromatography-mass spectrometry; all of the samples were assayed for the presence of drugs and changes in the drug concentrations both before and after preservation in formalin. Concentrations of assayed drugs decreased upon fixation in formalin; levels of these drugs did not necessarily show further decreases during subsequent storage in fixative, up to 13 months. Distinct trends in drug levels were found in liver and kidney. In liver, the levels of chlorpromazine, levomepromazine, and promethazine decreased to 23-39% at 1 month after preservation; all 3 of these drugs were detected at all tested time points of preservation. Bromazepam was not detected at 13 months after preservation. Milnacipran was the most unstable after preservation in formalin solution among all of the assayed drugs. In kidney, all assayed drugs exhibited reduced stability during preservation compared to levels in liver. Methamphetamine and methylenedioxymethamphetamine were not detected in any time points of tissues. The proportions of the drugs that remained within the tissues differed between liver and kidney. Also, S-oxide compounds of chlorpromazine and levomepromazine, which were not observed before preservation, were detected in fixed liver tissues and their fixatives at 3, 6 and 13 months of preservation. These results suggest that analyses in formalin-fixed tissues need to include analysis of various organ-tissues and their fixatives at multiple time points for the duration of preservation. These analyses should include detection of chemical degradation/denaturation products, such as S-oxides of chlorpromazine and levomepromazine.

Contribution of in utero drug exposure when interpreting hair results in young children.

Hair specimen is necessary to complement blood and/or urine analyses as it permits differentiation of a single exposure from chronic use of a drug by segmentation of the hair for a stated growth period. Moreover, due to a frequent long delay between event and police declaration, hair can be the only solution for lack of corroborative evidence of a committed crime. With the exception of lower amount of biological material in children versus adults, there is no specific analytical problem when processing samples from children. The issue is the interpretation of the findings, with respect to the different pharmacological parameters. In some very young children, the interpretation can be complicated by potential in utero exposure. Twenty-four cases from daily practice have been reviewed. Children were less than 1 year old, hair was always longer than 4 cm and the corresponding mothers admitted having used drugs during pregnancy. Drugs involved include methadone, tramadol, diphenhydramine, diazepam, cannabis, heroin, amitriptyline and bromazepam. Analyses were achieved by hyphenated chromatographic validated procedures after hair decontamination and segmentation. The concentrations measured in the hair of children were lower than those observed in subjects using therapeutically (or illegally) these drugs. In that sense, the frequency of exposures appears as un-frequent (low level of exposure), with marked decrease in the more recent period. However, the parents denied any administration in all cases and there was no reason to suspect re-exposure after delivery and no clinical problem during the period between delivery and hair collection during regular visits to the physician was noticed. The pattern of drug distribution was similar in all these cases, low concentrations in the proximal segments and highest concentration in the distal segment (last segment). When considering the concentration in the distal segment as the 100% of the response (highest concentration), after analysis of 4 segments (irrespective of the length of the segment but longer than 1cm), it was observed the following pattern: proximal segment, 5-35% of the response; segment 2, 15-50% of the response; segment 3, 25-60% of the response; and distal segment, 100% of the response. It is proposed to consider 100% in utero contribution to the final interpretation when the ratio concentration of the proximal segment to the concentration of the distal segment is lower than 0.5. This can be applied only when the child is under 1 year old and the hair shaft length is at least 4 cm (to achieve suitable segmentation). It is important, when using this cut-off to have at least 3 or 4 segments to be able to observe the variation in drug concentrations, whatever the length of each segment (>1cm).

Assisted suicide by fentanyl intoxication due to excessive transdermal application.

Herein, we report a case of an assisted suicide committed by application of 34 matrix-based fentanyl-containing transdermal therapeutic systems (TTS) with different release rates. The TTS were supplied by the husband but administered by the deceased herself. Besides routine systematic toxicological analysis (STA), the concentrations of fentanyl and norfentanyl were determined in the blood (femoral and heart), urine, stomach content, brain, lung tissue, musculus iliopsoas, liver, kidney, bile and in some of the used TTS by LC-MS/MS. Blood levels of fentanyl were 60.6 µg/L in femoral blood and 94.1 µg/L in heart blood. These concentrations are in good concordance with levels described in cases with accidental or lethal suicidal fentanyl patch application. The organ distribution indicates an influence of post-mortem redistribution. The levels of residual fentanyl in the TTS were also determined. STA furthermore revealed supratherapeutic levels of bromazepam. Thus, the cause of death was a combination of fentanyl and bromazepam intoxication. However, considering the determined levels of fentanyl and norfentanyl in the entire set of specimens and the high toxicity in comparison to bromazepam, fentanyl was the leading toxic noxa.

Segmental hair analysis can demonstrate external contamination in postmortem cases.

Excluding laboratory mistakes, a false positive hair result can be observed in case of contamination from environmental pollution (external contamination) or after drug incorporation into the hair from the individual body fluids, such as sweat or putrefactive fluid (post mortem artifact). From our 20 years experience of hair testing, it appears that artifact(s) cannot be excluded in some post mortem cases, despite a decontamination procedure. As a consequence, interpretation of the results is a challenge that deserves particular attention. Our strategy will be reviewed in this paper, based on six cases. In all cases, a decontamination procedure with two washes of 5 ml of dichloromethane for 5 min was performed and the last dichloromethane wash was negative for each target drug. From the histories, there was no suspicion of chronic drug use. In all six cases, the concentrations detected were similar along the hair shaft, irrespective of the tested segment. We have considered this as indicative of external contamination and suggested to the forces or the judges that it is not possible to indicate exposure before death. In contrast to smoke, it seems that contamination due to aqueous matrices (sweat, putrefactive fluid, blood) is much more difficult to remove. To explain potential incorporation of 7-aminoflunitrazepam via putrefactive material, the author incubated negative hair strands in blood spiked at 100 ng/ml and stored at +4°C, room temperature and +40 °C for 7, 14 and 28 days. After routine decontamination, 7-aminoflunitrazepam tested positive in hair, irrespective of the incubation temperature, as early as after 7 days (233-401 pg/mg). In all periods, maximum concentrations were observed after incubation at room temperature. The highest concentration (742 pg/mg) was observed after 28 days incubation at room temperature. It is concluded that a standard decontamination procedure is not able to completely remove external contamination in case of post mortem specimens. Homogenous segmental analyses can be probably indicative of external contamination and therefore a single hair result should not be used to discriminate long-term exposure to a drug. Nor should the presence of a metabolite be considered as a discrimination tool, as it can also be present in putrefactive material.

Environmental occurrence, fate and transformation of benzodiazepines in water treatment.

Benzodiazepine derivatives are prescribed in large quantities globally and are potentially new emerging environmental contaminants. Unfortunately, a dearth of data exists concerning occurrence, persistence and fate in the environment. This paper redresses this by reviewing existing literature, assessing the occurrence of selected benzodiazepine anxiolytics (diazepam, oxazepam and bromazepam) in wastewater influent and effluent and surface water from Slovenia, evaluating their removal during water treatment and identifying the transformation products formed during water treatment. Their occurrence was monitored in hospital effluent, river water and in wastewater treatment plant influent and effluent. The study reveals the presence of benzodiazepine derivatives in all samples with the highest amounts in hospital effluents: 111 ng L(-1), 158 ng L(-1) and 72 ng L(-1) for diazepam, bromazepam and oxazepam, respectively. Removal efficiencies with respect to biological treatment of diazepam were 16-18% (oxic), 18-32% (anoxic→oxic), 53-76% (oxic→anoxic) and 83% (oxic→anoxic→oxic→anoxic cascade bioreactors), while the removal oxazepam was 20-24% under anoxic conditions. Coupled biological and photochemical treatment followed by the adsorption to activated carbon resulted in a removal efficiency of 99.99%. Results reveal the recalcitrant nature of benzodiazepine derivatives and suggest that only combinational treatment is sufficient to remove them. In addition, eight novel diazepam and four novel oxazepam transformation products are reported.

Detection of amitriptyline, nortriptyline and bromazepam in liver, CSF and hair in the homicidal poisoning of a one-month-old girl autopsied 8 months after death.

We reported on the death by poisoning of a one-month-old baby that had followed the death of one of her sister (due to cyamemazine overdose). Exhumation of the corpse was done 8 months after burial and revealed the presence of amitriptyline. Parent drug and its metabolite were analysed by HPLC-MS/MS in positive ionisation mode on a C(18) analytical column using a gradient of acetonitrile and 2mM formate buffer at pH=3. Quantification is based on the main ion m/z=233, the common product ion of nortriptyline (MH(+), m/z 264), amitriptyline (MH(+), m/z 278) and nortriptyline D3 used as internal standard (MH(+), m/z 267). Amitriptyline and nortriptyline in the liver were measured at a concentration of 29.8 and 3.6 µg/g, respectively. Hair analyses revealed the presence of amitriptyline and nortriptyline at concentrations of 1811 and 43 pg/mg, respectively, while complementary analyses showed the presence of bromazepam in the hair at a concentration of 740 pg/mg, thus documenting previous administrations. The mother confessed later having used the drinkable form of the pharmaceutical LAROXYL(®) by pouring the content of a 20 ml bottle (at 40 mg/ml) into the feeding-bottle of her child. The milk was sweet but still bitter and following the testimony of a close relative, the whole family helped to feed the crying baby.

Spectrofluorimetric quantification of bromazepam using a highly selective optical probe based on Eu3+-bromazepam complex in pharmaceutical and serum samples.

A rapid, simple and sensitive spectrofluorimetric method for determination of trace amount of bromazepam is developed. In phosphate buffer of pH 7.4. The bromazepam enhance the luminescence intensity of the Eu(3+) ion in Eu(3+)-bromazepam complex at lambda(ex)=390nm. The produced luminescence intensity of Eu(3+)-bromazepam complex is in proportion to the concentration of bromazepam. The working range for the determination of bromazepam is 2.3x10(-8) to 6.2x10(-7)M with detection limit (LoD) and quantitative detection limit (LoQ) of 3x10(-9) and 1.2x10(-8)M, respectively. While, the working range, detection limit (LoD) and quantitative detection limit (LoQ) in case of the quantum yield calculations are 3.7x10(-8) to 3.4x10(-7)M with of 3.4x10(-9) and 9.2x10(-8)M, respectively. The enhancement mechanism of the luminescence intensity in the Eu(3+)-bromazepam system has been also explained.

Chemical submission: results of 4-year French inquiry.

Psychoactive substances may be administered without the knowledge of a victim in order to induce incapacitation and thus facilitate criminal actions. The characteristics of the victims and the drugs used in such suspected chemical submissions (CS) were analyzed in 309 cases collected from October 2003 to December 2007 through a national survey. Out of 309 cases, 158 met all criteria of CS. The victims were mostly female (n = 89, 56%). The type of aggression was mostly sexual assault (in 79 cases 50%). Benzodiazepines and related drugs were detected in 129 victims (82%) and were mostly clonazepam, zolpidem, and bromazepam whereas flunitrazepam and gamma hydroxybutyrate, well known for their use in CS, were identified in 11 (7%) and five (3%) of the 158 victims. CS is not an anecdotal phenomenon in France. Information for health professionals and workers in forensic structures as well as education of the general population associated with preventive measures such as drug dosage form changes should contribute to improved care management of victims and decreased risk.

Cardiac and peripheral blood similarities in the comparison of nordiazepam and bromazepam blood concentrations.

Concomitant heart and peripheral blood determinations were performed on 40 fatal cases involving nordiazepam (20 cases) and bromazepam (20 cases). The heart blood concentration for the two drugs (588 ng/mL for nordiazepam and 802 ng/mL for bromazepam) does not differ from the corresponding peripheral blood concentration (587 ng/mL for nordiazepam and 883 ng/mL for bromazepam). The mean ratios for the heart and peripheral blood concentrations were 0.95 for nordiazepam and 0.86 for bromazepam. No postmortem redistribution was observed for these two benzodiazepines. The authors thus suggest that corresponding heart blood can be proposed in the quantitative analysis of these drugs when peripheral blood is unavailable. The present study also shows the stability of the two drugs after a year of storage.

[Drug-facilitated crime and sexual abuse: a pediatric observation]. / Soumission chimique et agression sexuelle chez l'enfant: à propos d'une observation.

Drug-facilitated crime in sexual assault situations remains insufficiently recognized by physicians. In the possible context of an assault and in front of recent neuropsychicological disturbances in a child, such an issue has to be considered. The quality of sampling, the use of ultra-sensitive and specific toxicologic methods and a clinical-biological collaboration allow to recognize this form of delinquency whose consequences are both medical and legal.

Determination of bromazepam in human plasma by high-performance liquid chromatography with electrospray ionization tandem mass spectrometric detection: application to a bioequivalence study.

A simple method using a one-step liquid-liquid extraction (LLE) followed by high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometric (ESI-MS/MS) detection was developed for the determination of bromazepam in human plasma, using lorazepam as internal standard. The acquisition was performed in the multiple reaction monitoring mode, monitoring the transitions: m/z 316 > 182 for bromazepam and m/z 321 > 275 for lorazepam. The method was linear over the studied range (1-100 ng ml(-1)), with r(2) > 0.98, and the run time was 2.5 min. The intra- and inter-assay precisions were 2.7-14.6 and 4.1-17.3%, respectively and the intra- and inter-assay accuracies were 87-111 and 75.8-109.5%, respectively. The mean recovery was 73.7%, ranging from 64.5 to 79.7%. The limit of quantification was 1 ng ml(-1). At this concentration the mean intra- and inter-assay precisions were 14.6 and 7.1%, respectively, and the mean intra- and inter-assay accuracies were 102.5 and 104%, respectively. Bromazepam stability was evaluated and the results showed that the drug is stable in standard solution and in plasma samples under typical storage and processing conditions. The method was applied to a bioequivalence study in which 27 healthy adult volunteers (14 men) received single oral doses (6 mg) of reference and test bromazepam formulations, in an open, two-period, randomized, crossover protocol. The 90% confidence interval of the individual ratios (test formulation/reference formulation) for C(max) (peak plasma concentration), AUC(0-96) and AUC(0-inf) (area under the plasma concentration versus time curve from time zero to 96 h and to infinity, respectively) were within the range 80-125%, which supports the conclusion that the test formulation is bioequivalent to the reference formulation regarding the rate and extent of bromazepam absorption.

Determination of bromazepam, clonazepam and metabolites after a single intake in urine and hair by LC-MS/MS. Application to forensic cases of drug facilitated crimes.

The number of reports on drug facilitated crimes is increasing these last years. Apart from ethanol and cannabis, benzodiazepines (BZD) and analogs are the most common drugs reported to be used probably due to their amnesic and sedative properties. We have developed a rapid and sensitive method using LC-MS/MS triple stage quadrupole (TSQ) for the determination of single exposure to bromazepam (Lexomil, 6 mg) and clonazepam (Rivotril, 2 mg) in urine and hair of healthy volunteers. Chromatography was carried out on a Uptisphere ODB 5 microm, 2.1 mm x 150 mm column (Interchim) with a gradient of acetonitrile and formate 2 mM buffer, pH 3. Urine was extracted with Toxitube A (Varian) and allowed the detection of bromazepam, 3-hydroxy-bromazepam, clonazepam and 7-Aminoclonazepam for more than 6 days. Head hair, collected 1 month after the exposure, was treated by incubation with Soerensen buffer pH 7.6, followed by liquid-liquid extraction with dichloromethane for common BZD. A specific pre-treatment for amino-BZD, with an incubation of 15 min at 95 degrees C in 0.1 N NaOH before liquid-liquid extraction with dichloromethane, gave better recoveries and repeatability. After single exposure, bromazepam was present in powdered hair at 28 pg/mg and 7-Aminoclonazepam at 22 pg/mg in the first 1-cm segment, while no clonazepam was detectable. This method was applied in two forensic cases. It allowed us to determine bromazepam in urine 3 days after the alleged offense and in cut head hair at a concentration of 6.7 pg/mg only in the 2-cm proximal segment. The other case showed the presence of clonazepam and 7-Aminoclonazepam in urine a few hours after the offense and the presence of 7-Aminoclonazepam at about 3.2 pg/mg in axillary hair 4 months later.

Spectrophotometric and fluorimetric determination of diazepam, bromazepam and clonazepam in pharmaceutical and urine samples.

New spectrophotometric and fluorimetric methods have been developed to determine diazepam, bromazepam and clonazepam (1,4-benzodiazepines) in pure forms, pharmaceutical preparations and biological fluid. The new methods are based on measuring absorption or emission spectra in methanolic potassium hydroxide solution. Fluorimetric methods have proved selective with low detection limits, whereas photometric methods showed relatively high detection limits. Successive applications of developed methods for drugs determination in pharmaceutical preparations and urine samples were performed. Photometric methods gave linear calibration graphs in the ranges of 2.85-28.5, 0.316-3.16, and 0.316-3.16 microgml-1 with detection limits of 1.27, 0.08 and 0.13 microgml-1 for diazepam, bromazepam and clonazepam, respectively. Corresponding average errors of 2.60, 5.26 and 3.93 and relative standard deviations (R.S.D.s) of 2.79, 2.12 and 2.83, respectively, were obtained. Fluorimetric methods gave linear calibration graphs in the ranges of 0.03-0.34, 0.03-0.32 and 0.03-0.38 microgml-1 with detection limits of 7.13, 5.67 and 16.47 ngml-1 for diazepam, bromazepam and clonazepam, respectively. Corresponding average errors of 0.29, 4.33 and 5.42 and R.S.D.s of 1.27, 1.96 and 1.14 were obtained, respectively. Statistical Students t-test and F-test have been used and satisfactory results were obtained.

Acute fatal poisoning with cyamemazine.

A case of fatal poisoning with cyamemazine is presented. The cyamemazine was identified in post-mortem blood using a specific gas chromatographic/mass spectrometry method. The autopsy blood concentration of cyamemazine was 1800 ng/ml. Chronic use of cyamemazine was demonstrated by the presence of the drug in hair. Two other drugs were also detected (bromazepam and trimeprazine). We think that this current blood concentration (1800 ng/ml) is a fatal blood concentration because of the negativity of the other parameters, but careful interpretation of analytical findings are important, the possibility that this death was a consequence of the toxicity of combined drugs could not be excluded. Not many therapeutics and toxic levels were previously reported in overdosage cases in which cyamemazine was involved. We consider that this concentration is only of guidance value for a fatal cyamemazine poisoning.

Study on the inclusion complexes of bromazepam with beta- and beta-hydroxypropyl-cyclodextrins.

Solubility enhancement of the water insoluble bromazepam was studied during the formation of its inclusion complexes with beta-cyclodextrin (beta-CD) and beta-hydroxypropyl-cyclodextrin (beta-HP-CD). The phase solubility technique established by Higuchi and Connors and UV-spectrophotometric methods (zero- and second-order derivative approaches) were used to measure the changes introduced in this chemical system. The amount of time, which was necessary to reach equilibrium between inclusion complexes and their free components, was estimated and found equal to 24 h. The study was carried out at (i) pH 7.0 and 25 degrees C and (ii) pH 7.4 and 37 degrees C. The solubility of bromazepam increased linearly as a function of concentration for both beta-and beta-hydroxypropyl-cyclodextrins. Thus, the phase solubility diagrams were classified as of A(L) type in all cases. Under the above-mentioned conditions, the formation constants of the inclusion complexes were calculated and their stoichiometry was evaluated, found in the range of 69-85 M(-1) and 1:1, respectively.