The analysis of ramipril/ramiprilat concentration in human serum with liquid chromatography-tandem mass spectrometry - interpretation of high concentrations for the purposes of forensic toxicology.

Ramipril is a popular angiotensin-converting enzyme inhibitor applied in the treatment of hypertension. Its therapeutic effect is oriented on the concentration of the active metabolite ramiprilat. The information about toxic drug levels is missing in the literature. Therefore, the aim of this work was an indication of possible toxic ranges based on the analysis of real samples with high ramiprilat concentrations. For these purposes, an appropriate analytical LC-MS/MS method was developed and validated according to forensic guidelines and applied in the routine. Most real samples targeted for ramipril/ramiprilat were associated with the typical therapeutic drug range of 1-40 ng/mL described in the literature. However, higher drug levels with ramiprilat concentrations above 100 ng/mL could also be observed infrequently in cases of driving under the influence of drugs or attempted suicides. To the best of the author's knowledge, this is the first time antemortem ramipril and ramiprilat concentrations associated with driving under the influence of drugs and suicide attempts were discussed from a forensic point of view. The collected data enabled an indication of the ramiprilat toxic concentration range from about 600 ng/mL to at least 3500 ng/mL. The toxic concentration range discussed can be applied in the forensic practice as a reference for future cases.

Phenibut screening and quantification with liquid chromatography-tandem mass spectrometry and its application to driving cases.

An analytical strategy for identification by an LC-MS/MS multitarget screening method and a suitable LC-MS/MS based quantification were developed for the psychotropic drug phenibut. The samples analyzed were collected during traffic control and were associated with driving under the influence of drugs. A positive sample for phenibut was identified in a single case of driving under the influence. The quantification revealed a drug concentration of 1.9 µg/mL. An interaction with blood alcohol (BAC = 0.10%) was discussed as the explanation of the way of driving and deficit manifestations observed (swaying, nystagmus, quivering of the eyelid, and reddened eyes). According to the available information, the quantified phenibut concentration could be explained by an intake of four tablets (self-reported) during the day containing 250 mg of the drug. Chromatography was performed with a Luna 5 µm C18 (2) 100 A, 150 mm × 2 mm analytical column, and a buffer system consisted of 10 mM ammonium acetate and 0.1% acetic acid (v/v) included in mobile phases marked as A (H2 O/methanol = 95/5, v/v) and B (H2 O/methanol = 3/97, v/v). An effective limit of detection (LOD = 0.002 µg/mL) could be achieved for the multitarget screening method. The quantification of phenibut was performed on a second LC-MS/MS system with LOD/LOQ values of 0.22/0.40 µg/mL. Since phenibut quantification data are rare, the presented information can be used with caution for evaluation of positive cases in the future.

Antemortem and postmortem rodenticide analysis in forensic toxicology as a part of an LC-MS/MS-based multi-target screening strategy.

Since rodenticides represent a substance group relevant in toxicological analyses, the aim of this work was the development of a complex multi-target screening strategy for the identification with liquid chromatography-tandem mass spectrometry. A simple protein precipitation was used as the sample preparation strategy. Further, a Luna 5 µm C18 (2) 100 Å, 150 × 2 mm analytical column was applied for the separation of relevant analytes with a Shimadzu HPLC. Signal detection was performed with a SCIEX API 5500 QTrap MS/MS system. The rodenticides investigated (α-chloralose, brodifacoum, bromadiolone, coumatetralyl, difenacoum, and warfarin) could be incorporated effectively into a multi-target screening strategy covering about 250 substances representing different groups with a limit of detection appropriate for substance identification. The strategy can easily be modified to perform semi-quantitative measurements for this substance group and could be supplemented by quantification based on standard addition.

Detection of pharmaceuticals in "dirty sprite" using gas chromatography and mass spectrometry.

Dirty Sprite, also known as "lean" or "purple drank", is a preparation associated with the presence of codeine and promethazine. These drinks, predominantly used by young people, are mixtures of, for example, soft drinks, prescription medicines, and prescription cough syrups. The use of these illicit preparations started in Texas in the 1960s and become popularized in the 1990s. However, the misuse of these cocktails has become more common in other countries to date, for example, in Thailand. Given the illicit nature of these preparations and the lack of information available on the composition of these products, there is a need to identify and quantify the drugs that may be present. Three samples of Dirty Sprite were analyzed using GC-MS after liquid/liquid-extraction under acidic and basic conditions. Since the acidic extraction did not show the detection of relevant substances, samples were alkalized to pH ≥ 9, followed by extraction with 1-chlorobutane. GC-MS screening revealed the identification of codeine, dihydrocodeine, promethazine and impurities of cocaine. A selected ion monitoring method was developed for the quantification of these compounds using lemonade as a calibration matrix. Quantitative analysis showed concentrations of 130-mg/L codeine, 75-mg/L promethazine, and 3.4-mg/L cocaine in sample 1; 74-mg/L promethazine and 91-mg/L dihydrocodeine in sample 2; and 130-mg/L codeine combined with 68-mg/L promethazine in sample 3. The results also illustrate that the consumption of drugs detected in Dirty Sprite samples could lead to health risks given that these prescription medicines are consumed outside the medical environment.

Signal-Separated Quantification of γ-Hydroxybutyrate with Liquid Chromatography-Tandem Mass Spectrometry in Human Urine and Serum as an Improvement of the Analyte Adduct Ion-Based Quantification.

The aim of the work was the development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS-MS) γ-hydroxybutyrate (GHB) quantification method in urine and human serum by the use of the analyte adduct ion formation strategy. A combined detection with a conventional precursor ion in the negative electrospray mode and additionally GHB adduct ions with both sodium acetate and lithium acetate was in focus. Therefore, GHB quantification was based on separated MS-MS signals. Two tandem mass spectrometers representing different MS-MS generations (Sciex API 4000 QTrap and Sciex API 5500 QTrap) were used for method validation and comparison. Shimadzu HPLC systems equipped with a Luna 5-µm C18 (2) 100 A, 150-mm × 2-mm analytical column were successfully applied for sample analyses. Infusion experiments were performed for adduct identification and analyte detection optimization. Sample preparation could be limited to a simple and fast protein precipitation/sample dilution. An effective signal-separated GHB quantification with three independent precursor ions representing separated areas of the mass spectrum was developed, validated according to forensic guidelines and applied in the routine. The developed and applied strategy resulted in a higher safety factor for the analyte quantification performed in the forensic toxicology. A relevant analytical improvement could be achieved with this alternative adduct-based GHB analysis since a good correlation of analyte concentrations calculated on the basis of separated signals was stated as useful analytical information.

Practical aspect of dimer adduct formation in small-molecule drug analysis with LC-MS/MS.

Aim: Since the MS/MS based detection of small-molecule drugs with poor or even no ion fragmentation is a challenge in bioanalysis, alternative MS/MS detection strategies were in focus of this study and applied in the field of forensic toxicology. Material & methods: Analyte quantification with liquid chromatography-tandem mass spectrometry of problematic drugs was studied by the application of dimer adduct formation and valproic acid (VPA) was used as a model drug. VPA adduct ions could be identified during infusion experiments and the VPA dimer adduct ion was optimized for the detection. Conclusion: Dimer adduct ion formation can be used as an effective way of VPA quantification in human serum. Further, the parallel detection of dimer adduct ions with other adduct ion types can be stated as advantage in LC-MS/MS analysis of problematic drugs.

Interpretation of melperone intoxication: post-mortem concentration distribution and interpretation of intoxication data.

OBJECTIVES: Since melperone abuse with lethal intoxication is common, expert opinions based on therapeutical and lethal concentration ranges can be considered as important. Because there is a lack of information about fatalities caused by melperone mono-intoxications and data on tissue samples with concentration distribution, the aim of this work is the examination of lethal concentration ranges of melperone and drug quantification in different matrices. METHODS: An LC-MS/MS method was applied for analyses performed in blood and tissue samples. Quantification based on standard addition and sample preparation on liquid-liquid extraction with 1-chlorobutane. An appropriate tissue homogenization was performed ahead of extraction with an IKA Ultra-Turrax-Tube-Drive®. A Luna 5 µm C18 (2) 100 Å, 150  × 2 mm analytical column was used for chromatographic separation and the elution was performed with two mobile phases consisted of A (H2O/methanol = 95/5, v/v) and B (H2O/methanol = 3/97, v/v) both with 10 mM ammonium acetate and 0.1% acetic acid. RESULTS: A multi-drug LC-MS/MS analytical method developed was applied successfully for melperone quantification in different post-mortem matrices. No analytical problems could be identified during method development and analyses of real samples. The melperone lethal concentration calculated in femoral blood of the drug mono-intoxication investigated was 10 mg/L. Melperone concentration distribution was presented for the first time. CONCLUSIONS: The lethal reference concentration of melperone in femoral blood of 17.1 mg/L pointed out in different reference lists should be used with caution. Instead, a lower lethal melperone concentration should be considered. The post-mortem concentration distribution of the drug presented could be helpful in the interpretation of cases where no blood samples are available.

Interpretation of melperone intoxication: post-mortem concentration distribution and interpretation of intoxication data.

OBJECTIVES: Since melperone abuse with lethal intoxication is common, expert opinions based on therapeutical and lethal concentration ranges can be considered as important. Because there is a lack of information about fatalities caused by melperone mono-intoxications and data on tissue samples with concentration distribution, the aim of this work is the examination of lethal concentration ranges of melperone and drug quantification in different matrices. METHODS: An LC-MS/MS method was applied for analyses performed in blood and tissue samples. Quantification based on standard addition and sample preparation on liquid-liquid extraction with 1-chlorobutane. An appropriate tissue homogenization was performed ahead of extraction with an IKA Ultra-Turrax-Tube-Drive®. A Luna 5 µm C18 (2) 100 Å, 150  × 2 mm analytical column was used for chromatographic separation and the elution was performed with two mobile phases consisted of A (H2O/methanol = 95/5, v/v) and B (H2O/methanol = 3/97, v/v) both with 10 mM ammonium acetate and 0.1% acetic acid. RESULTS: A multi-drug LC-MS/MS analytical method developed was applied successfully for melperone quantification in different post-mortem matrices. No analytical problems could be identified during method development and analyses of real samples. The melperone lethal concentration calculated in femoral blood of the drug mono-intoxication investigated was 10 mg/L. Melperone concentration distribution was presented for the first time. CONCLUSIONS: The lethal reference concentration of melperone in femoral blood of 17.1 mg/L pointed out in different reference lists should be used with caution. Instead, a lower lethal melperone concentration should be considered. The post-mortem concentration distribution of the drug presented could be helpful in the interpretation of cases where no blood samples are available.

Determination of drugs in exhumed liver and brain tissue after over 9 years of burial by liquid chromatography-tandem mass spectrometry-Part 2: Benzodiazepines, opioids, and further drugs.

In this publication, benzodiazepines, opioids, and further drugs were analyzed in exhumed brain and liver tissue samples in 116 cases (total) after 9.5-16.5 years of burial. Solid phase extraction followed by liquid chromatography-tandem mass spectrometry was applied. Data from literature is listed summarizing the detectability of the presented analytes after a certain time of burial. In our study, 60% of the analyzed benzodiazepines, 100% of the opioids, and 82% of further drugs were detectable. Only the benzodiazepines lorazepam, nitrazepam, flunitrazepam, and its metabolite norflunitrazepam, and the drugs butylscopolamine, metronidazole, and omeprazole were not detectable at all. Percentage of positive findings (total, and separately for brain and liver tissue) and postmortem period are listed for each analyte. Correlation of detectability depending on postmortem period and condition of tissue are presented exemplarily for midazolam. No substantial correlation was observed. Despite a long time of burial, most benzodiazepines, opioids, and further drugs were detectable in the examined tissue samples. Our results may be a good support for future exhumations in which toxicological analyses are relevant.

Determination of drugs in exhumed liver and brain tissue after over 9 years of burial by liquid chromatography-tandem mass spectrometry-Part 1: Cardiovascular drugs.

This paper should serve as support for future exhumations in which an analysis of cardiovascular drugs is issued after over 9 years of burial. Amiodarone, amlodipine, atropine, bisoprolol, cafedrine, clonidine, esmolol, furosemide, hydrochlorothiazide, lisinopril, nifedipine, nitrendipine, phenprocoumon, torsemide verapamil, and xipamide were determined in liver and brain tissue of over 100 cases in which exhumation was performed after over 9 years of burial. Diagrams, showing the detectability depending on postmortem period as well as condition of tissues, are presented for furosemide.

Application of combined acetate salt based multiple analyte adduct formation in signal separated quantification performed for the purposes of forensic toxicology with liquid chromatography-tandem mass spectrometry - Discussion on the basis of salicylic acid applied as a model drug.

In this work the examination of analyte adduct formation by the application of both sodium acetate and lithium acetate was performed with salicylic acid used as a model drug. Additionally, on the basis of combined acetate salt based multiple analyte adduct ions it was aimed to investigate the generation of different analyte precursor ions and their application in signal separated quantification with liquid chromatography-tandem mass spectrometry. Adduct identification experiments were performed by infusion experiments and resulted in an optimised detection of analyte adduct ions with lithium acetate, sodium acetate and combined lithium acetate/sodium acetate. Sample preparation based on a fast protein precipitation with a methanol solution containing lithium acetate and sodium acetate. A Luna 5 µm C18 (2) 100 A, 150 mm × 2 mm analytical column and a mobile phase composed of an H2O/methanol = 3%/97% (v/v) solution with 10 mmol l-1 ammonium acetate and 0.1% acetic acid were used for chromatographic purposes. The linearity was investigated in the range of 5-500 µg/ml. The limit of detection (LOD of 1.2 µg/ml) and quantification (LOQ 4.0 µg/ml) together with other results of validation experiments revealed that this strategy can be applied for salicylic acid quantification.

The study and application of analyte adduct based ionisation of propofol in the analysis with liquid chromatography-tandem mass spectrometry.

The ionisation of propofol was studied on the basis of analyte adduct formation with the components of the mobile phase. The identification of an appropriate propofol adduct ion generated in an acetate salt based buffer in the negative electrospray ionisation mode made the propofol LC-MS/MS analysis with two multiple reaction monitoring experiments possible without analyte derivatisation. Since a product ion of the deprotonated analyte was also used in the detection, these two fragmentation experiments were performed with different precursor ions. Liquid-liquid extraction with 1-chlorobutane was applied for sample preparation. A Luna 5 µm C18 (2) 100 A, 150 mm × 2 mm analytical column together with a mobile phase composed of water/methanol (3/97, v/v) with 10 mmol·L-1 ammonium acetate and 0.1% acetic acid were applied. The limit of detection and quantification were 0.02 mg/L and 0.06 mg/L appropriate. Other results of the validation experiments performed like linearity of the calibration range, accuracy and precision, processed sample stability, recovery, matrix effect and selectivity revealed that this propofol quantification strategy developed can be defined as an applicable alternative to other methods presented.

Adduct Formation-Supported Two-Way Electrospray Ionization Strategy for the Determination of Urinary Creatinine Concentration with LC-MS-MS in Abstinence Control.

In this work, an alternative LC-MS-MS strategy for the analysis of urinary creatinine in abstinence control was presented and discussed. The two-way electrospray ionization consisted of two different precursor ions in which fragmentation was used in multiple reaction monitoring experiments. A creatinine adduct ion with sodium and sodium acetate together with the conventional analyte protonation was investigated. Adduct formation and fragmentation was explored by appropriate infusion experiments performed with analyte solutions prepared in different concentrations. The analytical signal was compensated by the application of appropriate isotopically labeled internal standard. The advantages of information carried by precursor ions separated in the mass spectra were pointed out. Sample preparation based solely on sample dilution performed in the final HPLC vial directly. A Luna 5 µm C18 (2) 100 A, 150 mm × 2 mm analytical column together with a mobile phase consisted of H2O/methanol = 3%/97% (v/v) with 10 mmol/L ammonium acetate and 0.1% acetic acid (flow = 0.4 mL/min) were used for the separation performed during a run of 5 min. The linearity was examined in the range of 100-3,000 mg/L. The limit of detection (13 mg/L), limit of quantification (43 mg/L) together with method precision/accuracy, selectivity, stability and matrix effect were tested to be appropriate for forensic applications. The applicability of water as surrogate matrix for method calibration was also examined successfully. The presented strategy was used in the analysis of real samples. No interferences with the creatinine peak eluted at about 1.0 min could be recorded.

Multiple analyte adduct formation in liquid chromatography-tandem mass spectrometry - Advantages and limitations in the analysis of biologically-related samples.

Multiple analyte adduct formation was examined and discussed in the context of reproducible signal detection in liquid chromatography-tandem mass spectrometry applied in the analysis of biologically-related samples. Appropriate infusion solutions were prepared in H2O/methanol (3/97, v/v) with 1 mM sodium acetate and 10 mM acetic acid. An API 4000 QTrap tandem mass spectrometer was used for experiments performed in the negative scan mode (-Q1 MS) and the negative enhanced product ion mode (-EPI). γ­Hydroxybutyrate and its deuterated form were used as model compounds to highlight both the complexity of adduct formation in popular mobile phases used and the effective signal compensation by the application of isotope-labelled analytes as internal standards.

Direct analysis of ethylene glycol in human serum on the basis of analyte adduct formation and liquid chromatography-tandem mass spectrometry.

The aim of this work was to develop a fast, cost-effective and time-saving liquid chromatography-tandem mass spectrometry (LC-MS/MS) analytical method for the analysis of ethylene glycol (EG) in human serum. For these purposes, the formation/fragmentation of an EG adduct ion with sodium and sodium acetate was applied in the positive electrospray mode for signal detection. Adduct identification was performed with appropriate infusion experiments based on analyte solutions prepared in different concentrations. Corresponding analyte adduct ions and adduct ion fragments could be identified both for EG and the deuterated internal standard (EG-D4). Protein precipitation was used as sample preparation. The analysis of the supernatant was performed with a Luna 5µm C18 (2) 100A, 150mm×2mm analytical column and a mobile phase consisting of 95% A (H2O/methanol=95/5, v/v) and 5% B (H2O/methanol=3/97, v/v), both with 10mmolL-1 ammonium acetate and 0.1% acetic acid. Method linearity was examined in the range of 100-4000µg/mL and the calculated limit of detection/quantification was 35/98µg/mL. However, on the basis of the signal to noise ratio, quantification was recommended at a limit of 300µg/mL. Additionally, the examined precision, accuracy, stability, selectivity and matrix effect demonstrated that the method is a practicable alternative for EG quantification in human serum. In comparison to other methods based on liquid chromatography, the strategy presented made for the first time the EG analysis without analyte derivatisation possible.

Imatinib quantification in human serum with LC-MS3 as an effective way of protein kinase inhibitor analysis in biological matrices.

BACKGROUND: As imatinib gained a lot of attention in the field of medicine, appropriate methods are needed for drug analysis. LC-MS/MS combined with complex sample preparation and column enrichment is usually the method of choice when high sensitivity is necessary. The application of LC-MS3 in imatinib quantification has not been discussed in the literature. METHODS: An LC-MS3 imatinib quantification method was developed and validated in human serum. The sample preparation was based on the liquid-liquid extraction of 50 µL human serum. Chromatographic separation was performed using a Luna 5 µm C18 (2) 100 A, 150 mm×2 mm column and the elution was done using a mobile phase consisting of A (H2O/methanol=95/5, v/v) and B (H2O/methanol=3/97, v/v), both with 10 mM ammonium acetate and 0.1% acetic acid. RESULTS: The conditions applied resulted in a limit of detection/quantification value of 0.14/0.45 ng/mL reached without a sophisticated sample preparation technique or enrichment column application. It could be demonstrated that MS3 detection is a very effective way of sensitive imatinib quantification. Further, it could be stated that the strategy presented can be very useful for a sensitive analysis of other protein kinase inhibitors, because their molecule structure is appropriate for MS3 detection. CONCLUSIONS: The presented analytical strategy is an effective way of protein kinase inhibitor analysis in human serum.