Magnetic Solid-Phase Microextraction Protocol Based on Didodecyldimethylammonium Bromide-Functionalized Nanoparticles for the Quantification of Epirubicin in Biological Matrices.

Due to epirubicin's (EPI) narrow therapeutic index and risk of cardiotoxicity, it is critical to monitor concentrations of this drug when being used to treat cancer patients. In this study, a simple and fast magnetic solid-phase microextraction (MSPME) protocol for the determination of EPI in plasma and urine samples is developed and tested. Experiments were performed using prepared Fe3O4-based nanoparticles coated with silica and a double-chain surfactant-namely, didodecyldimethylammonium bromide (DDAB)-as a magnetic sorbent. All the prepared samples were analyzed via liquid chromatography coupled with fluorescence detection (LC-FL). The validation parameters indicated good linearity in the range of 0.001-1 µg/mL with a correlation coefficient > 0.9996 for plasma samples, and in the range of 0.001-10 µg/mL with a correlation coefficient > 0.9997 for urine samples. The limit of detection (LOD) and limit of quantification (LOQ) for both matrices were estimated at 0.0005 µg/mL and 0.001 µg/mL, respectively. The analyte recovery after sample pretreatment was 80 ± 5% for the plasma samples and 90 ± 3% for the urine samples. The developed method's applicability for monitoring EPI concentrations was evaluated by employing it to analyze real plasma and urine samples collected from a pediatric cancer patient. The obtained results confirmed the proposed MSPME-based method's usefulness, and enabled the determination of the EPI concentration-time profile in the studied patient. The miniaturization of the sampling procedure, along with the significant reduction in pre-treatment steps, make the proposed protocol a promising alternative to routine approaches to monitoring EPI levels in clinical laboratories.

Profiling Docetaxel in Plasma and Urine Samples from a Pediatric Cancer Patient Using Ultrasound-Assisted Dispersive Liquid-Liquid Microextraction Combined with LC-MS/MS.

In recent years, therapeutic drug monitoring (TDM) has been applied in docetaxel (DOC)-based anticancer therapy to precisely control various pharmacokinetic parameters, including the concentration of DOC in biofluids (e.g., plasma or urine), its clearance, and its area under the curve (AUC). The ability to determine these values and to monitor DOC levels in biological samples depends on the availability of precise and accurate analytical methods that both enable fast and sensitive analysis and can be implemented in routine clinical practice. This paper presents a new method for isolating DOC from plasma and urine samples based on the coupling of microextraction and advanced liquid chromatography with tandem mass spectrometry (LC-MS/MS). In the proposed method, biological samples are prepared via ultrasound-assisted dispersive liquid-liquid microextraction (UA-DLLME) using ethanol (EtOH) and chloroform (Chl) as the desorption and extraction solvents, respectively. The proposed protocol was fully validated according to the Food and Drug Administration (FDA) and the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) requirements. The developed method was then applied to monitor the DOC profile in plasma and urine samples collected from a pediatric patient suffering from cardiac angiosarcoma (AS) with metastasis to lungs and mediastinal lymph nodes, who was receiving treatment with DOC at a dose of 30 mg/m2 body surface area. Due to the rarity of this disease, TDM was carried out to determine the exact levels of DOC at particular time points to ascertain which levels were conducive to maximizing the treatment's effectiveness while minimizing the drug's toxicity. To this end, the concentration-time profiles of DOC in the plasma and urine samples were determined, and the levels of DOC at specific time intervals up to 3 days after administration were measured. The results showed that DOC was present at higher concentrations in the plasma than in the urine samples, which is due to the fact that this drug is primarily metabolized in the liver and then eliminated with the bile. The obtained data provided information about the pharmacokinetic profile of DOC in pediatric patients with cardiac AS, which enabled the dose to be adjusted to achieve the optimal therapeutic regimen. The findings of this work demonstrate that the optimized method can be applied for the routine monitoring of DOC levels in plasma and urine samples as a part of pharmacotherapy in oncological patients.

Withania somnifera L.: Phenolic Compounds Composition and Biological Activity of Commercial Samples and Its Aqueous and Hydromethanolic Extracts.

In the present study, the chemical composition and bioactive properties of commercially available Withania somnifera samples were evaluated. The hydromethanolic and aqueous extracts of the tested samples were analyzed in terms of phenolic compound composition, ascorbic acid content, antioxidant and antibacterial activity, and acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities. Polyphenols and ascorbic acid content, as well as the antioxidant activity, were higher in the aqueous extracts than in the hydromethanolic extracts. Generally, aqueous extracts presented higher antioxidant activity than the hydromethanolic ones, especially in the case of 2,2-diphenyl-1-picryl-hydrazyl (DPPH) assay. Moreover, higher amounts of phenolic acids and flavonoids were found in the hydromethanolic extracts compared to the aqueous ones. Regarding the antibacterial properties, samples 4, 6, and 10 showed the best overall performance with growth-inhibitory activities against all the examined bacteria strains. Finally, the aqueous and hydromethanolic extracts were the most efficient extracts in terms of AChE and BChE inhibitory activities, respectively. In conclusion, our results indicate that W. somnifera possesses important bioactive properties which could be attributed to the high amounts of phenolic compounds. However, a great variability was recorded in commercially available products, suggesting significant differences in the origin of product and the processing method.

Simultaneous determination of mitotane, its metabolite, and five steroid hormones in urine samples by capillary electrophoresis using ß-CD2 SDS1 complexes as hydrophobic compounds solubilizers.

Mitotane is a cytotoxic drug used in the treatment of inoperable adrenocortical carcinoma, it inhibits steroidogenesis as well, and therefore monitoring the level of steroid hormones in patients treated with mitotane is a crucial point of therapy. Hence, we have developed a simple, fast, and efficient electrophoretic method combined with reverse polarity sweeping as online preconcentration technique and dispersive liquid-liquid microextraction for the simultaneous determination of mitotane, its main metabolite DDA, and five steroid hormones (progesterone, testosterone, epitestosterone, cortisol, and corticosterone) in urine samples. In addition, a new sample matrix consisting of ß-CD2 SDS1 complexes for a high hydrophobic compounds solubilization was developed. Approach based on the application of ß-cyclodextrin and SDS complex of a ratio 2:1 allowed for hydrodynamic injection into the capillary of a solution containing both mitotane and other analytes. The detection limits of the analytes for the reverse polarity sweeping-dispersive liquid-liquid microextraction method were found to be in the range of 1.5-3 ng/mL, which were approximately 1000 times lower than in the conventional hydrodynamic injection (5 s, 0.5 psi) without any preconcentration procedure. All analytes were completely resolved in less than 13 min by uncoated silica capillary with an inner diameter of 75 µm (ID) × 60 cm. Electrophoretic separation was performed in reverse polarity with a voltage of -25 kV with a background electrolyte (BGE) consisting of 100 mM SDS, 25% ACN, 25 mM phosphate buffer (pH 2.5), and 7 mM ß-cyclodextrin.

Sensitive Analysis of Idarubicin in Human Urine and Plasma by Liquid Chromatography with Fluorescence Detection: An Application in Drug Monitoring.

A new approach for the sensitive, robust and rapid determination of idarubicin (IDA) in human plasma and urine samples based on liquid chromatography with fluorescence detection (LC-FL) was developed. Satisfactory chromatographic separation of the analyte after solid-phase extraction (SPE) was performed on a Discovery HS C18 analytical column using a mixture of acetonitrile and 0.1% formic acid in water as the mobile phase in isocratic mode. IDA and daunorubicin hydrochloride used as an internal standard (I.S.) were monitored at the excitation and emission wavelengths of 487 and 547 nm, respectively. The method was validated according to the FDA and ICH guidelines. The linearity was confirmed in the range of 0.1-50 ng/mL and 0.25-200 ng/mL, while the limit of detection (LOD) was 0.05 and 0.125 ng/mL in plasma and urine samples, respectively. The developed LC-FL method was successfully applied for drug determinations in human plasma and urine after oral administration of IDA at a dose of 10 mg to a patient with highly advanced alveolar rhabdomyosarcoma (RMA). Moreover, the potential exposure to IDA present in both fluids for healthcare workers and the caregivers of patients has been evaluated. The present LC-FL method can be a useful tool in pharmacokinetic and clinical investigations, in the monitoring of chemotherapy containing IDA, as well as for sensitive and reliable IDA quantitation in biological fluids.

Development and validation of a high-performance liquid chromatographic method with a fluorescence detector for the analysis of epirubicin in human urine and plasma, and its application in drug monitoring.

The aim of the work was to develop a simple, sensitive and accurate liquid chromatography with fluorescence detection (LC-FL) method for the determination of epirubicin in human urine and plasma. Solid phase extraction with HLB cartridges and mixture of dichloromethane:2-propanol:methanol (2:1:1, v/v/v) as the eluent, was used to prepare the samples. The chromatographic analysis was carried out on a Synergi Hydro-RP column with a mobile phase consisting of 40 mM phosphate buffer (pH 4.1) and acetonitrile (69:31, v/v). Epirubicin was monitored at 497 nm and 557 nm for excitation and emission wavelengths, respectively. Validation data confirmed that the limit of detection and limit of quantification was 0.25 ng/mL and 0.5 ng/mL in both matrices. Next, the optimized LC-FL method was applied to determine the level of epirubicin in real samples taken from a 19-year-old patient with metastatic alveolar rhabdomyosarcoma (RMA) to create a drug profile. Plasma and urine samples were collected for 24 h after the end of a 6-hour infusion of epirubicin. The obtained results confirmed that the optimized and validated LC-FL method can be successfully used in drug monitoring therapy, pharmacokinetic and clinical studies. Moreover, the current work is also drawing attention to the relatively high level of epirubicin in the patient urine, which requires compliance with the safety rules in contact with this biological fluid by both medical staff and others, e.g. family members.

Determination of twenty pharmaceutical contaminants in soil using ultrasound-assisted extraction with gas chromatography-mass spectrometric detection.

In this paper, an analytical method for the simultaneous determination of twenty pharmaceuticals (eight non-steroidal anti-inflammatory drugs, five oestrogenic hormones, two antiepileptic drugs, two ß-blockers, and three antidepressants) in soils was developed. The optimal method included ultrasound-assisted extraction, a clean-up step on a silica gel column, derivatization using N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and 1% trimethylchlorosilane (TMCS) in pyridine and ethyl acetate (2:1:1, v/v/v) for 30 min at 60 °C, and determination by gas chromatography-mass spectrometry working in the selected ion monitoring mode. This affords good resolution, high sensitivity and reproducibility, and freedom from interferences even from complex matrices such as soils. The method detection limits ranged from 0.3 to 1.7 ng g-1, the intra-day precision represented as RSDs ranged from 1.1 to 10.0%, and the intra-day accuracy from 81.3 to 119.7%. The absolute recoveries of the target compounds were above 80%, except for valproic acid and diethylstilbestrol. The developed method was successfully applied in the analysis of the target compounds in soils collected in Poland. Among the 20 pharmaceuticals, 12 compounds were detected at least once in the soils. The determination of antiepileptic drugs, ß-blockers, and antidepressants was also performed for the first time.

Application of SPME supported by ionic liquids for the determination of biogenic amines by MEKC in clinical practice.

The analysis of biogenic amines (BAs) and their metabolites is helpful for the diagnosis of central nervous system disorders and other neuroendocrine and cancer disturbances. In the study, a developed micellar electrokinetic chromatography method, coupled with diode array detection (MEKC-DAD), was validated to monitor levels of adrenaline (A), noradrenaline (NA), dopamine (DA), L-Tryptophan (L-Tryp) and L-Tyrosine (L-Tyr) in real human urine samples. These neurotransmitters were isolated from urine samples using solid-phase microextraction (SPME) and methanol containing 1-ethyl-3-methylimidazolium tetrafluoroborate ionic liquid as the desorption phase. The method was linear for DA, A and L-Tyr in the range of 0.5-20 µg/mL and for NA and L-Tryp in the range of 0.25-20 µg/mL. The good linearity for BAs was confirmed by the correlation coefficient (R2) from 0.9989 for A to 0.9997 for NA and L-Tryp, respectively. The validation assays for accuracy, precision, limit of detection, limit of quantification, absolute recovery, and stability of the analytes were consistent with the requirements recommended by the FDA and ICH guidelines. Next, the validated SPME-MEKC method was successfully used for the quantification of A, NA, DA, L-Tryp and L-Tyr in real human urine samples collected from pediatric patients suffering from neuroblastoma, ganglioneuroblastoma, Wilms' tumor, rhabdoid tumor and lipoblastomatosis, as well as from healthy volunteers. Finally, the levels of BAs in cancer patients were evaluated as to whether they can be used as biomarkers of various health disturbances.

Recent Trends in the Quantification of Biogenic Amines in Biofluids as Biomarkers of Various Disorders: A Review.

Biogenic amines (BAs) are bioactive endogenous compounds which play a significant physiological role in many cell processes like cell proliferation and differentiation, signal transduction and membrane stability. Likewise, they are important in the regulation of body temperature, the increase/decrease of blood pressure or intake of nutrition, as well as in the synthesis of nucleic acids and proteins, hormones and alkaloids. Additionally, it was confirmed that these compounds can be considered as useful biomarkers for the diagnosis, therapy and prognosis of several neuroendocrine and cardiovascular disorders, including neuroendocrine tumours (NET), schizophrenia and Parkinson's Disease. Due to the fact that BAs are chemically unstable, light-sensitive and possess a high tendency for spontaneous oxidation and decomposition at high pH values, their determination is a real challenge. Moreover, their concentrations in biological matrices are extremely low. These issues make the measurement of BA levels in biological matrices problematic and the application of reliable bioanalytical methods for the extraction and determination of these molecules is needed. This article presents an overview of the most recent trends in the quantification of BAs in human samples with a special focus on liquid chromatography (LC), gas chromatography (GC) and capillary electrophoresis (CE) techniques. Thus, new approaches and technical possibilities applied in these methodologies for the assessment of BA profiles in human samples and the priorities for future research are reported and critically discussed. Moreover, the most important applications of LC, GC and CE in pharmacology, psychology, oncology and clinical endocrinology in the area of the analysis of BAs for the diagnosis, follow-up and monitoring of the therapy of various health disorders are presented and critically evaluated.

Optimization of LC method for the quantification of doxorubicin in plasma and urine samples in view of pharmacokinetic, biomedical and drug monitoring therapy studies.

A simple, rapid, reliable and sensitive method based on liquid chromatography with fluorescence detection (LC-FL) for the quantification of doxorubicin (DOX) in human plasma and urine samples was developed. The assay was carried out after the solid-phase extraction procedure (SPE) with hydrophilic-lipophilic balance (HLB) cartridges, and with daunorubicin hydrochloride (DAU) used as the internal standard. Chromatographic separation was performed on a Discovery HS C18 column in isocratic elution mode, and the detection of the analytes set at excitation and emission wavelengths of 487 and 555 nm, respectively. The developed LC-FL method has been validated for accuracy, precision, selectivity, linearity, recovery and stability. The limits of detection and quantification for DOX were 0.5 and 1 ng/mL in both biological fluids, respectively. Linearity was confirmed in the range of 1-1000 ng/mL and 0.001-25 µg/mL in plasma and urine samples, respectively, with a correlation coefficient greater than 0.9994. The proposed LC-FL method is selective, precise and accurate, and has been successfully applied for drug monitoring in pediatric cancer patients treated with DOX as a component of OEPA (Oncovin (Vincristine)-Etoposide-Prednisone-Adriamycin) and IOA (Ifosfamide-Oncovin-Adriamycin) chemotherapeutic schemes. Moreover, real exposure of hospital personnel to the anthracycline drugs in plasma and urine was evaluated in clinical practice.

Determination of urinary biogenic amines' biomarker profile in neuroblastoma and pheochromocytoma patients by MEKC method with preceding dispersive liquid-liquid microextraction.

The unbalanced secretion of biogenic amines (BAs) is considered to be a relevant biochemical biomarker in the screening for neuroendocrine tumors, such as: neuroblastoma and pheochromocytoma. However, there is still a need to improve the bioanalytical procedures for BA determination in biological samples due to their instability (photo- and thermosensitivity, easy oxidation) and low concentration in the body fluids. In this study, the primary analytical challenge was to optimize the method of extraction of seven compounds from among BAs and their precursors from urine samples. Several methods based on liquid-liquid extraction (LLE) or solid phase extraction (SPE) techniques were tested. By optimization of the extraction and data analysis using chemometric tool, the dispersive liquid-liquid microextraction (DLLME) has been chosen due to its low solvents consumption, high efficiency of isolation, preconcentration and suitable clean-up of biological matrix. Further, α-cyclodextrin-modified micellar electrokinetic chromatography (MEKC) with ultraviolet detection (UV) has been applied for quantification of the analyzed biologically active compounds with limits of detection (LOD) and limits of quantification (LOQ) at 0.15 and 0.5µgmL-1, respectively. Finally, the optimized and validated DLLME-MEKC-UV method has been employed for the analysis of real urine samples, obtained from 6 children with neuroendocrine tumors and 6 healthy children. It was stated that concentrations of BA could serve to differentiate between the patients and healthy children. This pilot study indicates that the elaborated fast and sensitive DLLME-MEKC-UV method for determination of panel of biomarkers could be successfully applied in everyday clinical practice to help to confirm the clinical diagnosis of neuroendocrine tumors in children.

Determination of Bendamustine in Human Plasma and Urine by LC-FL Methods: Application in a Drug Monitoring.

Simple and sensitive liquid chromatography (LC) methods with fluorescence (FL) detection for the determination of bendamustine (BM) in human plasma and urine were developed and validated. The procedure of BM extraction from a plasma sample involved solid-phase extraction with a C18 SPE column, while liquid-liquid extraction with dichloromethane was applied for a urine sample. In both methods, cinoxacin was used as the internal standard. Chromatographic separations were performed on a Synergi Max-RP column, while FL detector was set at the excitation wavelength of 328 nm and the emission wavelength of 420 nm. The LC-FL methods were validated for accuracy, precision, selectivity, linearity, recovery, and stability. The detection limits for BM were 0.5 and 2.5 ng mL-1 in plasma and urine, respectively. The intra-day and inter-day precisions were less than 9.86 %, while the accuracies were higher than 92.63 and 94.29 % for BM in plasma and urine, respectively. The proposed LC-FL methods were sensitive, robust, and specific, allowing reliable drug quantification in plasma and urine samples. The methodologies were successfully applied to monitoring of BM in a child with cancer treated with BM.

Cyclodextrin-modified MEKC method for quantification of selected acidic metabolites of catecholamines in the presence of various biogenic amines. Application to diagnosis of neuroblastoma.

The main aim of the presented study was to develop a reliable and non-time-consuming method for the simultaneous separation of biogenic amines (BAs) like noradrenalin, adrenalin, dopamine and their main metabolites - homovanillic acid (HVA), vanillylmandelic acid (VMA), 3,4-dihydroxyphenylacetic acid (DOPAC) - in urine samples. To achieve this, the validated α-cyclodextrin (α-CD)-modified micellar electrokinetic chromatography method with DAD was proposed. The optimized separation parameters were as follows: background electrolyte was composed of 10mM sodium tetraborate decahydrate, 30mM SDS, 15% (v/v) methanol and 25mM α-CD, adjusted to pH 9.36 with 1N NaOH; uncoated fused silica capillary (75µm i.d.×60.2cm length); λ=200nm; injection time 5s, applied voltage 25kV; temperature 25 (±0.1)°C. Next, the developed MEKC method was practically applied to evaluate the levels of selected acidic metabolites of catecholamines like HVA, VMA and DOPAC in urine samples collected from patients diagnosed with neuroblastoma (NB), melanotic neuroectodermal tumor of infancy (MNTI).

Capillary electromigration techniques as tools for assessing the status of vitamins A, C and E in patients with cystic fibrosis.

The purpose of this work is the evaluation of the nutritional status of patients with cystic fibrosis (CF), based on the level of vitamin C in urine and vitamins A and E in serum, using the fast, selective and fully automated micellar electrokinetic capillary chromatographic (MEKC) and microemulsion electrokinetic capillary chromatographic (MEEKC) methods. The optimization of parameters affecting the electrophoretic separation provided adequate separation of the analytes of interest in the short time of 8 min (MEKC) and 20 min (MEEKC). The developed methods were practical applications to evaluate the levels of vitamins A, C and E in real samples from 28 children suffering from cystic fibrosis and from 10 healthy volunteers. Based on the mean concentration values obtained in the two groups, it can be seen that the levels of each vitamin were lower in patients with CF than in healthy volunteers. In the case of vitamin E, these differences in both groups were statistically significant, while the disproportion of concentrations of vitamins A and C in both the studied groups were not so relevant. On the other hand, a principal component analysis (PCA) confirmed that in some patients with CF the concentration of vitamin A was significantly lower than in the control group. Thus, the future evaluation of the status of fat-soluble vitamins in the longer term for the evaluation of the nutritional status of patients with CF should be continued. The presented CE methods can become useful tools for the evaluation of the nutritional status of patients with CF.

Gel electrophoretic separation of proteins from cultured neuroendocrine tumor cell lines.

Neuroendocrine tumors (NET) often develop asymptomatically and are detected at a late stage. Currently, there exist certain markers of NET that occur only in the advanced stages of the disease. Still, there is need to develop markers specific of the early stage of cancer development. Nevertheless, biomarkers are mostly low­abundant proteins and require separation from complex protein mixtures, which remains a major challenge. The goal of the present study was to optimize one­dimensional­polyacrylamide gel electrophoresis (1D­PAGE) for separation and comparison of protein composition from neuroendocrine tumor samples. 1D­PAGE was optimized by modification of the gel concentration and by comparison of different gel staining protocols. In addition, several steps prior to electrophoresis were carried out to purify and preliminarily reduce the complexity of the sample. The results of these optimization steps indicated that use of an albumin removal kit can considerably decrease the amount of albumin in the samples, thereby allowing to detect proteins of low abundance. Optimal separation of the sample was obtained using a 12% polyacrylamide gel. Furthermore, the use of silver staining allowed detection of proteins at nanogram levels, whereas for Coomassie Brilliant Blue staining, the detection limit was 10 times higher. Optimization of the sample preparation workflow and parameters of the electrophoretic separation allowed to reduce the complexity of the studied material and facilitated further identification of proteins of low abundance in the sample. This study demonstrated that analysis of the secreted proteome of NET cells by 1D­PAGE is a simple and suitable tool for the identification of potential NET protein biomarkers.

Chemometric evaluation of urinary steroid hormone levels as potential biomarkers of neuroendocrine tumors.

Neuroendocrine tumors (NETs) are uncommon tumors which can secrete specific hormone products such as peptides, biogenic amines and hormones. So far, the diagnosis of NETs has been difficult because most NET markers are not specific for a given tumor and none of the NET markers can be used to fulfil the criteria of high specificity and high sensitivity for the screening procedure. However, by combining the measurements of different NET markers, they become highly sensitive and specific diagnostic tests. The aim of the work was to identify whether urinary steroid hormones can be identified as potential new biomarkers of NETs, which could be used as prognostic and clinical course monitoring factors. Thus, a rapid and sensitive reversed-phase high-performance liquid chromatographic method (RP-HPLC) with UV detection has been developed for the determination of cortisol, cortisone, corticosterone, testosterone, epitestosterone and progesterone in human urine. The method has been validated for accuracy, precision, selectivity, linearity, recovery and stability. The limits of detection and quantification were 0.5 and 1 ng mL-1 for each steroid hormone, respectively. Linearity was confirmed within a range of 1-300 ng mL-1 with a correlation coefficient greater than 0.9995 for all analytes. The described method was successfully applied for the quantification of six endogenous steroid levels in human urine. Studies were performed on 20 healthy volunteers and 19 patients with NETs. Next, for better understanding of tumor biology in NETs and for checking whether steroid hormones can be used as potential biomarkers of NETs, a chemometric analysis of urinary steroid hormone levels in both data sets was performed.

Chemometric optimization of derivatization reactions prior to gas chromatography-mass spectrometry analysis.

Derivatization is a methodological technique that can be used to make an organic compound more suitable for qualitative and/or quantitative analysis (e.g. pharmaceuticals). However, many analysts try to avoid this analytical procedure as it is time-consuming and labour-intensive. On the other hand, an inter-laboratory study demonstrated that with regard to sensitivity and measurement uncertainty, gas chromatography coupled to mass-spectrometry was superior to liquid chromatography coupled to mass spectrometry for the trace analysis of organic compounds in matrices of greater complexity. In our previous paper (Kumirska et al., J. Chemometr. 25 (2011) 636-643) we suggested using principal component analysis (PCA) to optimize the derivatization of six compounds (5 oestrogenic steroids and diethylstilbestrol) prior to GC-MS analysis. In the present work we applied a highly sophisticated model - 24 pharmaceuticals derived from six classes of drugs. The efficiency of different derivatization reactions was evaluated by PCA and compared with that obtained from cluster analysis (CA), the latter method being applied in this context for the first time. Derivatization using N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and 1% trimethylchlorosilane (TMCS) in pyridine and ethyl acetate (2:1:1, v/v/v) for 30min at 60°C was found to be optimal. The SPE-GC-MS method was also validated and successfully applied to the analysis of selected pharmaceuticals in wastewater and surface waters in Poland.