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.

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.

Reconsideration of sample pH adjustment in bioanalytical liquid-liquid extraction of ionisable compounds.

Liquid-liquid extraction (LLE) is widely used as a simple and robust sample preparation technique in bioanalytical sample preparation. When extracting ionisable compounds, most bioanalysts adjust the pH of the sample to achieve fully unionized compounds. Usually, a generally accepted rule is applied to adjust the pH of the aqueous phase, known as the pKa+/-2 rule, depending on the acid/base characteristics of the analyte. By taking a closer look at the general equations that describe the extraction behaviour of ionisable compounds, we extended this pH adjustment rule by taking the distribution ratio and the volume of both liquid phases into account. By choosing an extraction pH based on this extended rule, the selectivity of the extraction can be influenced without loss of recovery. As a measure of this selectivity, two equations were proposed to indicate the ability of the extraction system to discriminate between two compounds. Also, milder extraction pH can be used for pH labile analytes. To use this new rule quantitatively, a new calculation method for the determination of the distribution ratio was derived. These calculations were based on normalized recoveries making this method less susceptible to errors in absolute recovery determination. The proposed equations were supported by demonstrating that careful pH adjustment can lead to higher selectivity. The main conclusion was that a closer look at the extraction pH in bioanalytical methods extends the possibilities of obtaining a higher selectivity or the possibilities of extracting pH labile analytes at milder pH conditions without loss of recovery.

New practical algorithm for modelling retention times in gradient reversed-phase high-performance liquid chromatography.

Computer models have been widely used to predict the chromatographic behaviour of liquid chromatography systems. With the introduction of mass spectrometric detection and the use of lower mobile phase flow rates with conventional LC equipment, the influence of the dwell volume on the shape of the gradient curve becomes an issue in predicting the retention times. A new straight forward algorithm is proposed for the modelling of retention times in reversed-phase LC, taking the effect of the dwell volume on the gradient shape into account. The results show that the dwell volume has a large effect at lower flow rates and on the retention times of the analytes eluting at the end of fast gradient curves. The proposed model is able to make reliable predictions and can be helpful in LC-MS method development.

The detection and identification of quaternary nitrogen muscle relaxants in biological fluids and tissues by ion-trap LC-ESI-MS.

Quaternary nitrogen muscle relaxants pancuronium, rocuronium, vecuronium, gallamine, suxamethonium, mivacurium, and atracurium and its metabolites were extracted from whole blood and other biological fluids and tissues by using a solid-phase extraction procedure. The extracts were examined by using high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). The drugs were separated on a ODS column in a gradient of ammonium acetate buffer (pH 5.0) and acetonitrile. Full-scan mass spectra of the compounds showed molecular ions, and MS-MS spectra showed fragments typical of the particular compounds. LC-ESI-MS allowed an unequivocal differentiation of all muscle relaxants involved. The method was applied in a case of rocuronium and suxamethonium administration in a Caesarian section and in a case of intoxication by pancuronium injection. In both cases, the administered drugs could be detected and identified in the supplied samples.

Feasibility of the direct coupling of solid-phase extraction-pipette tips with a programmed-temperature vaporiser for gas chromatographic analysis of drugs in plasma.

Solid-phase extraction-pipette tips (SPE-PTs) were used for micro solid-phase extraction of lidocaine and diazepam from plasma. Off-line extraction was followed by on-line desorption. On-line desorption was carried out by direct coupling of the SPE-PTs with the liner of the programmed-temperature vaporiser. This coupling only required shortening of the liner by maximally 16 mm, cutting the SPE-PT, and equipping the remaining part with two O-rings. Due to the heating of the injector the SPE-PTs were heated as well, which resulted in a significant amount of impurities. Pre-heating and pre-washing was performed prior to the extraction to reduce the impurity level. The internal coupling device was applied successfully for the analysis of plasma samples with gas chromatography (GC) and mass-selective detection. Detection limits of 0.75 ng/ml and 2.5 ng/ml were obtained for lidocaine and diazepam, respectively, using 200 microl plasma. Recoveries for both compounds were about 80%. Although it is possible, the internal coupling device was not developed to be used as such. The main goal of this coupling was to show the feasibility of the integration of SPE-PTs with GC and to realize an important step to new automated SPE-GC systems.

Dynamically coated capillaries improve the identification power of capillary zone electrophoresis for basic drugs in toxicological analysis.

In systematic toxicological analysis (STA), analytical methods should have a high identification power. This can be suitably expressed by parameters such as mean list length (MLL) or discriminating power (DP). The reproducibility of a method has a great impact on its identification power, and should be as high as possible. In this study, two separation methods based on capillary zone electrophoresis (CZE) were evaluated towards STA applications. Besides a normal phosphate buffer, the commercially available buffer CElixir was used, which is a double-layer dynamic coating system. The coating stabilizes the endoosmotic flow, is independent of the pH, and is claimed to be more reproducible and faster at low pH than with normal buffers. A test set of 73 basic pharmaceutical compounds was analyzed by the two CZE methods. The total analysis time, including rinsing steps, was 8 min when the coating was used and 18 min without the coating. Effective mobilities were calculated and the reproducibilities were a factor of 2 better when the coating was used (between-days SD 0.020 and 0.040 m2/V s with and without the coating, respectively). MLL and DP were calculated for the two CZE methods and for combinations with standardized liquid and gas chromatography systems. CZE with CElixir coating clearly has a high potential for STA applications, as it was shown to have a higher identification power and shorter analysis times than normal CZE.

Screening for the presence of drugs in serum and urine using different separation modes of capillary electrophoresis.

Capillary electrophoresis (CE) is a modern separation technique that has some distinct advantages for toxicological analysis, such as a high efficiency, fast analysis, flexibility, and complementary separation mechanisms to chromatographic methods. CE can be applied in various modes, which each have a different separation mechanism or selectivity. The most common mode is capillary zone electrophoresis (CZE), in which charged analytes migrate in a buffer under the influence of an electric field. In micellar electrokinetic chromatography (MEKC), micelles are added to the buffer which interact with the analytes. MEKC can also be used for the separation of neutral compounds. In non-aqueous CE (NACE), the aqueous buffer is replaced by a background of electrolytes in organic solvents. A sample that needs to be screened can easily be analyzed subsequently by these CE modes using the same instrumentation. The aim of the study was to develop procedures for the analysis of basic and acidic drugs in serum and urine using CZE, MEKC, and NACE. A test mixture that consisted of six basic and six acidic compounds was used to study the separation behavior of five CE methods. The results showed that three methods (based on CZE, MEKC, and NACE) were suitable for the analysis of basic compounds and three methods (based on CZE and MEKC) for the analysis of acidic compounds. For the extraction of analytes from serum and urine, a solid-phase extraction (SPE) and a liquid-liquid extraction (LLE) method were compared. Both SPE and LLE methods provided clean extracts after extraction of the basic compounds from serum and urine. The extracts of acidic compounds contained more matrix interferences, especially for urine. The SPE method had some advantages compared to LLE, as it lead to cleaner extracts and higher peaks, and as it elutes basic and acidic compounds in one fraction. The potentials and pitfalls of the various methods for screening purposes in analytical toxicology are discussed.

Adsorptive voltametry to determine platinum levels in plasma from testicular cancer patients treated with cisplatin.

Patients cured of metastatic testicular cancer with cisplatin chemotherapy may suffer late adverse effects even after 20 years. The cause of these late adverse effects has not been elucidated yet. One cause might be prolonged tissue retention of platinum in these patients. Therefore, an extremely sensitive method for measuring platinum in plasma was used to investigate whether platinum is still detectable in plasma 10 to 20 years after cisplatin chemotherapy. High-pressure decomposition of plasma is followed by adsorptive voltametric determination of platinum, with a limit of quantification of 6 pg/g plasma. This procedure appeared suitable for the measurement of platinum in 44 former patients with platinum levels ranging from 22 to 140 pg/g plasma. This method is approximately 6000 times more sensitive than the standard flameless atomic absorption spectrophotometry (AAS) method. The platinum levels of these 44 patients were significantly elevated when compared with 20 control patients who were cured of testicular cancer but did not receive cisplatin chemotherapy (p < 0.001). There was a significant correlation between plasma platinum concentrations and follow-up time after cisplatin administration (r = -0.658, p < 0.001). This study shows that patients with testicular cancer who were treated with cisplatin can retain platinum in their body for at least 20 years. More data are needed to investigate whether there is a relation between the prolonged retention of platinum and long-term toxicity.

Intra- and interinstrument reproducibility of migration parameters in capillary electrophoresis for substance identification in systematic toxicological analysis.

The intra- and interinstrument reproducibilities of four capillary electrophoresis instruments were studied for identification purposes in systematic toxicological analysis (STA). A test set of 20 acidic test compounds and 5 reference compounds were analyzed for five days on each instrument using capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). The buffers consisted of 90 mM borate set at pH 8.4 (CZE) and 20 mM phosphate and 50 mM sodium dodecyl sulfate set at pH 7.5 (MEKC). All analyses were carried out using fused silica capillaries at an electric field strength of 52.6 kV/m. The use of a reproducible identification parameter is very important in STA. To deal with the poor reproducibility of the migration time, we recently introduced the corrected effective mobility. In this study, we investigated the intra- and interinstrument reproducibility of the migration time, the effective mobility, and the corrected effective mobility. Large differences in intra-instrument reproducibility were found when the migration time was used. The calculation of the effective mobility and the corrected effective mobility diminished these differences and enhanced the interinstrument reproducibility roughly by a factor 3. For (corrected) effective mobilities, intrainstrument reproducibilities were between 0.8-2.6% and interinstrument reproducibilities were between 3.2-3.9%.

How to approach substance identification in qualitative bioanalysis.

The ultimate goal in qualitative analysis in the biosciences is to demonstrate with acceptable probability that for an unknown constituent in a sample only one substance comes into consideration and that all other substances can be rejected. In the biosciences, identification of relevant substances in complex matrices through database retrieval is frequently required. Yet, despite its importance, the subject has not received much attention, so that progress has been limited and relevant literature is scarce. As a result, one can conclude from many publications and reports that qualitative analysis in practice is often not being addressed properly. In this paper, some fundamental aspects of qualitative analysis will be discussed and a general approach is provided for the correct identification of organic substances in complex matrices through database retrieval. Special attention is given to the choice of proper analytical techniques and their inter-laboratory standard deviations, as well as to match factors and decision criteria based on applying multiple analytical techniques, also if the latter have different dimensions (e.g. retention data and spectral data). In addition, the requirements for suitable databases are outlined and the need for inter-laboratory cooperation is emphasized.

SPEC disc solid-phase extraction for rapid broad-spectrum drug screening in urine.

Broad-spectrum drug screening requires that all relevant substances be isolated, detected, and identified, regardless of their structure and/or polarity. To this end, systematic solid-phase extraction (SPE) approaches for drug isolation from biological fluids are required. Because speed and cost effectiveness are key issues in analytical toxicology, we have evaluated a disc-format extraction device for this purpose and compared the latter with an existing packed-bed column-format method. The discs were SPEC.PLUS.C18AR/MP3 cartridges with 10-mL solvent reservoirs, providing hydrophobic and cation exchange interactions. Blank human urine was spiked at 2 microg/mL with a selection of acidic, neutral, and basic drugs representing a variety of relevant drug classes. Urine specimens (2 mL) were diluted with 2 mL 0.1 M phosphate buffer (pH 5.0) and then applied to the preconditioned disc. Washing was done with 1 mL water. Acidic and neutral drugs were eluted with 1 mL ethyl acetate/acetone (1:1), and basic drugs were eluted with 1 mL ammoniated ethyl acetate. The eluates were collected separately, evaporated down to about 0.1 mL, and analyzed by gas chromatography-flame-ionization detection to check cleanliness, recoveries, and reproducibilities. The discs showed good extraction properties for all drugs and were easy to handle. Recoveries were 75-100% with coefficients of variation of around 5%. The resulting eluates showed only a few matrix interferences. As compared to our standard SPE method with packed-bed columns, the disc procedure allowed reductions in elution volumes and total processing time of approximately 60-65%.

Rapid extraction of clenbuterol from human and calf urine using empore C8 extraction disks.

In the present paper, disk extraction was evaluated for the rapid isolation of clenbuterol from human and calf urine, followed by high-performance liquid chromatography analysis with UV detection. A method was developed for the extraction with standard density C8 disks. The disks could be washed with 25% methanol in 0.01M sodium hydroxide without significant losses of clenbuterol. The recovery of denbuterol was about 85%, and the extracts were clean. The detection limit was about 10 ng/mL. The main advantages of these disks were the saving of time and the reduced amounts of organic solvents needed.

Evaluation of the programmed temperature vaporiser for large-volume injection of biological samples in gas chromatography.

The use of a programmed temperature vaporiser (PTV) with a packed liner was evaluated for the injection of large volumes (up to 100 microl) of plasma extracts in a gas chromatograph. Solvent purity, which is essential when large volumes are injected into the GC system, was determined. Special attention was paid to the purity of the solvents used for the solid-phase extraction (SPE) procedure. For this SPE method, ethyl acetate was used as the extraction and reconstitution solvent, and thus the purity of the ethyl acetate was critical, especially when a non-selective GC detector was applied. The liquid capacity and inertness of different packed liners were investigated. The liner packed with ATAS "A" (modified Chromosorb-based material with special treatment) was found to be the most suitable for the analysis of the tested drugs. Good linearity in response for variations in volume and concentration was observed. A comparison was made between the applicability of flame ionisation detection (FID) and mass-selective detection (MSD). When 50-microl volumes of plasma extracts were injected with the PTV, the detection limits for secobarbital, lidocaine, phenobarbital and diazepam were about 50-times lower than when 1-microl volumes were injected. The detection limits of the tested compounds in plasma for injection of 50-100 microl plasma extract are 5-10 ng/ml for GC-FID whereas plasma concentrations of 250 pg/ml can be detected using the selected ion monitoring (SIM) mode of a MSD. For non-selective GC-FID, the background from a 50-microl injection was substantially larger than with 1-microl injection as a result of co-injected plasma matrix components and solvent impurities. These background effects were less with GC-MSD in the total ion current mode and virtually absent with GC-MSD in the SIM mode.

Evaluation of capillary electrophoretic techniques towards systematic toxicological analysis.

Two capillary electrophoresis (CE) methods were evaluated for their suitability in systematic toxicological analysis (STA). A test set of 25 barbiturates was analysed using capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). Buffers used consisted of 90 mM borate set at pH 8.4 (CZE) and 20 mM phosphate, 50 mM sodium dodecyl sulphate set at pH 7.5 (MEKC). All analyses were carried out using fused silica capillaries using an electric field strength of 52.6 kV/m. The use of a reproducible identification parameter is very important in STA as it influences the identification power (IP). To deal with the poor reproducibility of the migration time, we introduced the corrected effective mobility. Inter-day reproducibilities of the latter parameter were < 0.6% for CZE and < 0.5% for MEKC, using daily prepared buffers. The IP of the methods was expressed by calculation of the discriminating power and the mean list length. Data obtained were compared to gas chromatographic and high-performance liquid chromatographic data, and correlations between all methods were calculated. It was shown that little correlation exists between chromatographic and electrophoretic techniques. The results indicated that CE has a good identification power for the application in STA, especially when a combination of methods having a low correlation is used.

Multiresidue analysis of beta2-agonist in human and calf urine using multimodal solid-phase extraction and high-performance liquid chromatography with electrochemical detection.

Various beta2-agonists are used as illegal growth promoters in man and in animals. We developed a multiresidue procedure for the analysis of four beta-agonists in human and calf urine. The sample was pre-extracted with an Extrelut column at alkaline pH. The beta-agonists were eluted with a mixture of tert.-butylmethyl ether and hexane. Then the extract was further cleaned with a mixed mode SPE column, or with a combination of immunoaffinity chromatography (IAC) and the mixed mode SPE column. The IAC column contained antibodies against salbutamol, which were suitable for multiresidue extractions. The extract was then brought onto a mixed mode SPE column at an acidic pH. The column was washed with 70% methanol in water. Thereafter, the beta-agonists were eluted with ammoniated ethanol-hexane. The extract was analysed with an HPLC method with electrochemical detection. The beta-agonists were separated on a reversed-phase column using a mobile phase buffered at pH 5.5 and containing an ion-pair reagent. Recoveries were higher when the IAC procedure was not performed (90-105% vs. 65-75%), but the extracts were cleaner when the latter step was included. Detection limits in human and calf urine were in the low ng/ml range. The study indicated that beta2-agonists can be analysed in human and calf urine without the selectivity of a mass spectrometer, but that comprehensive clean-up is required to avoid the interference of urine matrix components.

Multi-residue analysis of anabolics in calf urine using high-performance liquid chromatography with diode-array detection.

We describe the development of an HPLC method with diode-array detection (DAD) for the analysis and identification of 20 substances with anabolic properties, that are considered as potential growth promoters, to be used for the analysis of extracts of calf urine samples. The substances are separated on an RP-Select B column using a mobile phase consisting of a mixture of acetonitrile and water. Gradient elution from 43-76% acetonitrile in water with a concave curve was used to achieve a good separation of the compounds with an acceptable analysis time. For the identification, a retention parameter and the UV spectrum were used. The retention parameter was the retention time corrected with a reference mixture. The latter reduced the standard deviations to about 25% of their original values. The limits of detection of the HPLC system ranged from 0.5-5 ng injected amount for the androgens, progestagens, stilbenes and resorcylic acid lactones and to 5-10 ng injected amount for the oestrogens. After extraction from urine the limits of detection were increased by the presence of matrix components, but they were between 5 and 10 ng injected amount for most of the substances.

Solid-phase extraction procedures in systematic toxicological analysis.

In systematic toxicological analysis (STA) the substance(s) present is (are) not known at the start of the analysis. In such an undirected search the extraction procedure cannot be directed to a given substance but must be a general procedure where a compromise must be reached in that the substances of interest are isolated at a yield as high as possible and the interfering substances from the biological material are removed. When using solid-phase extraction (SPE) it is desirable to have procedures using just one column. An overview of screening procedures using diatomaceous earth, polystyrene-divinylbenzene copolymer and mixed-mode bonded silica as column material in SPE is given. The latter type of sorbent is most popular at the moment and the critical steps in the procedure are outlined in more detail. Recent developments of SPE disks look very promising for STA.

Direct enantiomeric separation of mianserin and 6-azamianserin derivatives using chiral stationary phases.

The direct enantiomeric separation of mianserin and 6-azamianserin and some of their derivatives, respectively, by means of HPLC using two different chiral selectors was investigated. For the cellulose-based Chiralcel OD column, a strong dependence of the lipophilicity of the compounds tested on the retention behaviour was observed. To some extent, this was also found for the enantiomeric separation on the amylose-based Chiralpak AD column. In some cases a complementary behaviour of these two phases was observed: racemic mixtures that could not be separated by one column could be resolved by the other one.

Dramatic signal reduction in ion-mobility spectrometry by residues of solvents.

During our evaluation of ion-mobility spectrometry (IMS) screening for the abuse of the beta-agonist clenbuterol during fattening in cattle, we found that clenbuterol could not be detected with the instrument in extracts obtained by solid-phase extraction or ion-pair extraction, although the recovery for these procedures was at least 85%. Several experiments showed that the signal loss occurred mainly during the evaporation of the organic solvent. Again, it could be shown that no clenbuterol was lost during this step. On further investigation, we found that so-called solvent residues, which are left after the evaporation of the organic solvent, caused a significant reduction of the clenbuterol signal. Other reagents used in our sample pretreatment procedures were also found to reduce the signal. For example, the signal reductions caused by the different reagents used for the ion-pair extraction of clenbuterol from human urine were determined. We think that IMS is only useful for our purpose if a chromatographic preseparation is performed.