Journal of Chromatography B (v.962, #C)

The toxicity of the sea mango (Cerbera manghas L.) is well known. The plant is ranked as one of the deadliest of the southern Asian coastline. Cardenolidic heterosides are responsible for the cardiotoxicity of trees of the Cerbera genus. We have identified and determined the concentration of the principal glycosidic steroids present in the seeds of sea mangos (Thailand). Drug screening of an extract of the seeds was carried out using ultra-high performance liquid chromatography coupled to photodiode array detection and mass spectrometry (UHPLC–PDA-MS) with quantification at 219 nm. Identification was confirmed by UHPLC–HRMS. Deacetyltanghinin (m/z 549.3055 ± 2 ppm), neriifolin (m/z 535.3259 ± 2 ppm), tanghinin (m/z 591.3169 ± 2 ppm) and cerberin (577.3375 ± 2 ppm) were the most abundant glycosidic steroids present in the sea mango seeds. A seed of the dried ripe fruit had concentrations of 1209.1, 804.2, 621.4 and 285.9 μg/g, respectively. A seed of the fresh unripe fruit had concentrations of 49.4, 47.0, 3.5 and 2.3 μg/g.
Keywords: Cerbera manghas L.; Cardenolides; Forensic toxicology; Ultra-high performance liquid chromatography–photodiode array-mass spectrometry (UHPLC–PDA-MS); Ultra-high performance liquid chromatography–high resolution tandem mass spectrometry (UHPLC–HRMS/MS);

Quantification of folate metabolites in serum using ultraperformance liquid chromatography tandem mass spectrometry by Xiuwei Wang; Ting Zhang; Xin Zhao; Zhen Guan; Zhen Wang; Zhiqiang Zhu; Qiu Xie; Jianhua Wang; Bo Niu (9-13).
Folate deficiency is considered a risk factor for many diseases such as cancer, congenital heart disease and neural tube defects (NTDs). There is a pressing need for more methods of detecting folate and its main metabolites in the human body. Here, we developed a simple, fast and sensitive ultraperformance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) method for the simultaneous quantifications of folate metabolites including folic acid, 5-methyltetrahydrofolate (5-MeTHF), 5-formyltetrahydrofolate (5-FoTHF), homocysteine (Hcy), S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH). The method was validated by determining the linearity (r 2  > 0.998), sensitivity (limit of detection ranged from 0.05 to 0.200 ng/mL), intra- and inter-day precision (both CV < 6%) and recovery (each analyte was >90%). The total analysis time was 7 min. Serum samples of NTD-affected pregnancies and controls from a NTD high-risk area in China were analyzed by this method, the NTD serum samples showed lower concentrations of 5-MeTHF (P  < 0.05) and 5-FoTHF (P  < 0.05), and higher concentrations of Hcy (P  < 0.05) and SAH (P  < 0.05) compared with serum samples from controls, consistent with a previous study. These results showed that the method is sensitive and reliable for simultaneous determination of six metabolites, which might indicate potential risk factors for NTDs, aid early diagnosis and provide more insights into the pathogenesis of NTDs.
Keywords: UPLC/MS/MS; Neural tube defects; Folic acid; One-carbon metabolism;

A specific and reliable HPLC-MS/MS method was developed and validated for the simultaneous determination of 1-β-d-Arabinofuranosylcytosine (ara-C), 1-β-d-Arabinofuranosyluracil (ara-U) and 1-β-d-Arabinofuranosylcytosine triphosphate (ara-CTP) in the leukemic cells for the first time. The analytes were separated on a C18 column (100 mm × 2.1 mm, 1.8 μm) and a triple-quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source was used for detection. The ion-pairing reagent, NFPA, was added to the mobile phase to retain the analytes in the column. The cell homogenates sample was prepared by the simple protein precipitation. The calibration curves were linear over a concentration range of 3.45–3450.0 ng/mL for ara-C, 1.12–1120.0 ng/mL for ara-U and 4.13–4130.0 ng/mL for ara-CTP. The intra-day and inter-day precision was less than 15% and the relative error (RE) were all within ±15%. The validated method was successfully applied to assess the disposition characteristics of ara-C and support cell pharmacokinetics after the patients with leukemia were intravenously infused with SDAC and HiDAC. The result of the present study would provide the valuable information for the ara-C therapy.
Keywords: HPLC-MS/MS; Ara-C; Ara-U; Ara-CTP; Cell pharmacokinetics;

A simple, sensitive and reliable analytical method was developed for the simultaneous determination of 38 veterinary drugs (18 sulfonamides, 11 quinolones and 9 benzimidazoles) and 8 metabolites of benzimidazoles in bovine milk by ultra high performance liquid chromatography-positive electrospray ionization tandem mass spectrometry (UHPLC–ESI-MS/MS). Samples were extracted with acidified acetonitrile, cleaned up with Oasis® MCX cartridges, and analyzed by LC–MS/MS on an Acquity UPLC® BEH C18 column with gradient elution. The method allows such multi-analyte measurements within a 13 min runtime while the specificity is ensured through the MRM acquisition mode. The method was validated according to the European Commission Decision 2002/657/EC determining specificity, decision limit (CCα), detection capability (CCβ), recovery, precision, linearity and stability. For compounds which have MRLs in bovine milk, the CCα values fall into a range from 11 to 115 μg/kg, and the CCβ values fall within a range of 12–125 μg/kg. For compounds which have not MRLs in bovine milk, the CCα values fall into a range from 0.01 to 0.08 μg/kg, and the CCβ values fall within a range of 0.02–0.11 μg/kg. The mean recoveries of the 46 analytes were between 87 and 119%. The calculated RSD values of repeatability and within-laboratory reproducibility experiments were below 11% and 15% for the 46 compounds, respectively. The method was demonstrated to be suitable for the simultaneous determination of sulfonamides, quinolones and benzimidazoles in bovine milk.
Keywords: Sulfonamides; Quinolones; Benzimidazoles; Milk; LC–MS/MS;

Protocol for simultaneous isolation of three important banana allergens by Jasna Nikolic; Ivan Mrkic; Milica Grozdanovic; Milica Popovic; Arnd Petersen; Uta Jappe; Marija Gavrovic-Jankulovic (30-36).
Banana fruit (Musa acuminata) has become an important food allergen source in recent years. So far, 5 IgE reactive banana proteins have been identified, and the major allergens are: Mus a 2 (a class I chitinase, 31 kDa), Mus a 4 (thaumatin-like protein, 21 kDa), and Mus a 5 (β-1,3-glucanase, 33 kDa). Due to variations in allergen expression levels, diagnostic reagents for food allergy can be improved by using individual allergen components instead of banana allergen extracts. The purpose of this study was to optimize the purification protocol of the three major allergens present in banana fruit: Mus a 2, Mus a 4 and Mus a 5. By employing a three-step purification protocol (a combination of anion-exchange, cation-exchange and reversed-phase chromatography) three important banana allergens were obtained in sufficient yield and high purity. Characterization of the purified proteins was performed by both biochemical (2-D PAGE, mass fingerprint and N-terminal sequencing) and immunochemical (immunoblot) methods. IgE reactivity to the purified allergens was tested by employing sera of five allergic patients. The purified allergens displayed higher sensitivity in IgE detection than the routinely used extracts. The three purified allergens are good candidates for reagents in component-based diagnosis of banana allergy.
Keywords: Allergens; Purification; Banana; Chitinase; TLP; Glucanase;

A rapid, sensitive and selective UPLC/MS method using LTQ Orbitrap mass spectrometry was established for the analysis and characterization of the main biological components and their metabolites in rat plasma, urine and feces following oral administration of Lithocarpus polystachyus extract. In vivo, 22 flavonoid metabolites were observed in rat plasma, and 13 metabolites were detected in rat urine, whereas just two aglycones of dihydrochalcone (3-hydroxy phloretin and phloretin) could be detected in rat feces. Among these metabolites, one new and a known dihydrochalcone metabolite were isolated and definitely identified. Besides, five dihydrochalcone metabolites were tentatively identified as new compounds. A metabolism study of 3-hydroxyphlorizin and phloridzin was also conducted. Glucuronidation was the main metabolic pathway of phloridzin, whereas glucuronidation and sulfonation were the main metabolic pathway of 3-hydroxyphlorizin. These results provided a basis for evaluating the bioactive components of a complex natural medicine and their mechanisms of actions.
Keywords: Lithocarpus polystachyus; Biological components; Metabolites; UPLC–MS/MS;

The enantioseparation of ten mandelic acid derivatives was performed by reverse phase high performance liquid chromatography with hydroxypropyl-β-cyclodextrin (HP-β-CD) or sulfobutyl ether-β-cyclodextrin (SBE-β-CD) as chiral mobile phase additives, in which inclusion complex formations between cyclodextrins and enantiomers were evaluated. The effects of various factors such as the composition of mobile phase, concentration of cyclodextrins and column temperature on retention and enantioselectivity were studied. The peak resolutions and retention time of the enantiomers were strongly affected by the pH, the organic modifier and the type of β-cyclodextrin in the mobile phase, while the concentration of buffer solution and temperature had a relatively low effect on resolutions. Enantioseparations were successfully achieved on a Shimpack CLC-ODS column (150 × 4.6 mm i.d., 5 μm). The mobile phase was a mixture of acetonitrile and 0.10 mol L−1 of phosphate buffer at pH 2.68 containing 20 mmol L−1 of HP-β-CD or SBE-β-CD. Semi-preparative enantioseparation of about 10 mg of α-cyclohexylmandelic acid and α-cyclopentylmandelic acid were established individually. Cyclodextrin-enantiomer complex stoichiometries as well as binding constants were investigated. Results showed that stoichiometries for all the inclusion complex of cyclodextrin-enantiomers were 1:1.
Keywords: Mandelic acid derivatives; Chiral separation; Hydroxypropyl-β-cyclodextrin; Sulfobutyl ether-β-cyclodextrin; High performance liquid chromatography;

Two types of automated solid phase extraction (SPE) were assessed for the determination of human exposure to fentanyls in urine. High sensitivity is required to detect these compounds following exposure because of the low dose required for therapeutic effect and the rapid clearance from the body for these compounds. To achieve this sensitivity, two acceptable methods for the detection of human exposure to seven fentanyl analogs and three metabolites were developed using either off-line 96-well plate SPE or on-line SPE. Each system offers different advantages: off-line 96-well plate SPE allows for high throughput analysis of many samples, which is needed for large sample numbers, while on-line SPE removes almost all analyst manipulation of the samples, minimizing the analyst time needed for sample preparation. Both sample preparations were coupled with reversed phase liquid chromatography and isotope dilution tandem mass spectrometry (LC–MS/MS) for analyte detection. For both methods, the resulting precision was within 15%, the accuracy within 25%, and the sensitivity was comparable with the limits of detection ranging from 0.002 ng/mL to 0.041 ng/mL. Additionally, matrix effects were substantially decreased from previous reports for both extraction protocols. The results of this comparison showed that both methods were acceptable for the detection of exposures to fentanyl analogs and metabolites in urine.
Keywords: Fentanyls; Fentanyl; Solid phase extraction; Liquid chromatography; Mass spectrometry; Method development; Automation;

A fast analytical strategy of second-order calibration method based on the alternating trilinear decomposition algorithm (ATLD)-assisted high performance liquid chromatography coupled with a diode array detector (HPLC-DAD) was established for the simultaneous determination of eight flavonoids (rutin, quercetin, luteolin, kaempferol, isorhamnetin, apigenin, galangin and chrysin) in propolis capsules samples. The chromatographic separation was implemented on a Wondasil™ C18 column (250 mm × 4.6 mm, 5 μm) within 13 min with a binary mobile phase composed of water with 1% formic acid and methanol at a flow rate of 1.0 mL min−1 after flavonoids were only extracted with methanol by ultrasound extraction for 15 min. The baseline problem was overcome by considering background drift as additional compositions or factors as well as the target analytes, and ATLD was employed to handle the overlapping peaks from analytes of interest or from analytes and co-eluting matrix compounds. The linearity was good with the correlation coefficients no less than 0.9947; the limit of detections (LODs) within the range of 3.39–33.05 ng mL−1 were low enough; the accuracy was confirmed by the recoveries ranged from 91.9% to 110.2% and the root-mean-square-error of predictions (RMSEPs) less than 1.1 μg/mL. The results indicated that the chromatographic method with the aid of ATLD is efficient, sensitive and cost-effective and can realize the resolution and accurate quantification of flavonoids even in the presence of interferences, thus providing an alternative method for accurate quantification of analytes especially when the complete separation is not easily accomplished. The method was successfully applied to propolis capsules samples and the satisfactory results were obtained.
Keywords: Propolis; Flavonoids; HPLC-DAD; ATLD; Second-order advantage;

Validation of a UHPLC–MS/MS method for quantification of zearalenone, α-zearalenol, β-zearalenol, α-zearalanol, β-zearalanol and zearalanone in human urine by H. Belhassen; I. Jiménez-Díaz; R. Ghali; H. Ghorbel; J.M. Molina-Molina; N. Olea; A. Hedili (68-74).
Humans can be exposed to mycotoxins through the diet. Evaluation of exposure levels to mycotoxins can be performed by direct determination in urine. The present work proposes a sensitive ultrahigh-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method for the determination of zearalenone (ZON) and its five metabolites (α-zearalenol [α-ZOL], β-zearalenol [β-ZOL], α-zearalanol [zeranol, α-ZAL], β-zearalanol [teranol, β-ZAL] and zearalanone [ZAN]) in human urine samples. The method involves the enzymatic hydrolysis of the samples, extraction of the analytes using liquid–liquid extraction (LLE) with ethyl acetate/formic acid (99:1 v/v) and a cleanup step using hexane, prior to their quantification by UHPLC–MS/MS, using an electrospray ionization (ESI) interface in the negative mode. Zearalenone-d6 (ZON-d6) was used as surrogate. The limits of detection and the limits of quantification ranged from 0.03 to 0.3 ng mL−1 and from 0.1 to 1.0 ng mL−1, respectively. The method was validated using matrix-matched calibration and a spike recovery assay. Recovery rates for spiked samples ranged from 96% to 104%, with relative standard deviations lower than 8.5%. This method was satisfactorily applied to 42 urine samples from Tunisian women for the determination of zearalenone and its five metabolites.
Keywords: Mycotoxins; Zearalenone; Zearalenone metabolites; UHPLC–MS/MS; Human urine samples; Tunisian women;

In this present study, a sensitive and rapid UPLC–MS/MS method was developed for simultaneous quantification of paeoniflorin, albiflorin, ferulic acid, tetrahydropalmatine, protopine, typhaneoside and senkyunolide I in Beagle dog plasma after oral administration of the Shao-Fu-Zhu-Yu Decoction. Chloramphenicol and clarithromycin were used as internal standards. Plasma samples were processed by protein precipitation with methanol. The separation was performed on an Acquity BEH C18 column (100 mm × 2.1 mm, 1.7 μm) at a flow-rate of 0.4 mL/min, using 0.1% formic acid–acetonitrile as mobile phase. Method validation was performed as per Food and Drug Administration guidelines and the results met the acceptance criteria. After validation, this method was successfully applied to a pharmacokinetic study. The results showed that the apparent plasma clearance of paeoniflorin, albiflorin, typhaneoside and senkyunolide I were significantly higher than others. Double peak was observed in plasma concentration curves of tetrahydropalmatine, the ferulic acid had a good absorption in Beagle dog plasma, and senkyunolide I was detected in plasma from the first blood sampling time (15 min) and rapidly reached T max. The compound of typhaneoside has a low bioavailability according to the results.
Keywords: Shaofu Zhuyu decoction; UPLC–MS/MS; Pharmacokinetics; Beagle dog plasma;

Therapeutic drug monitoring (TDM) of antiepileptic drugs (AEDs) has been recognized as a useful tool in management of epilepsy. We developed a simple analytical method for simultaneous determination of four second generation AEDs, including gabapentin (GBP), pregabalin (PGB), vigabatrin (VGB), and topiramate (TOP). Analytes were extracted from human plasma using universal solid phase extraction, derivatized with 4-chloro-7-nitrobenzofurazan (NBD-Cl) and analyzed by HPLC with fluorescence detection. Using mass spectrometry we confirmed that NBD-Cl reacts with sulfamate group of TOP similarly as with amine group of the other three analytes. The method is linear (r 2  > 0.998) across investigated analytical ranges (0.375–30.0 μg/mL for GBP, PGB, and VGB; 0.50–20.0 μg/mL for TOP). Intraday and interday precision do not exceed 9.40%. The accuracy is from 95.6% to 106%. The recovery is higher than 80.6%, and the lower limit of quantification is at least 0.5 μg/mL. The method is selective and robust. For TOP determination the method was compared to a previously published method and the results obtained by the two methods were in good agreement. The developed method is suitable for routine TDM.
Keywords: Gabapentin; Pregabalin; Vigabatrin; Topiramate; Fluorescence detection; Therapeutic drug monitoring;

Separation of human immunoglobulin G subclasses on a protein A monolith column by Pelin Leblebici; M. Enis Leblebici; Frederico Ferreira-da-Silva; Alírio E. Rodrigues; Luís S. Pais (89-93).
Monolithic columns have attracted significant attention for the purification of large biomolecules. In the present study, a step gradient elution method was evaluated for the separation of human immunoglobulin G (hIgG) into its subclasses on CIM (convective interaction media) r-protein A (recombinant protein A) monolithic column. hIgG was loaded onto the column and bound protein was eluted with a pH gradient. The subclass content of the eluted fractions was analyzed by enzyme-linked immunosorbent assay (ELISA). Results showed that separation of IgG3 from the other three subclasses can be successfully achieved with high selectivity (100%) and throughput on monolithic media. It was also revealed that enriched fractions of IgG1 and IgG2 could be obtained from purified hIgG in a 28 min long chromatographic run. Three fractions with high IgG1 content (89.1%, 94.3% and 88.8%) were recovered. Furthermore, IgG2 was enriched to 64% successfully. A rapid step gradient elution scheme without any additives in buffers was proven to obtain enriched preparations of the two important subclasses with high throughput. The separation time can be reduced even more by increasing the flow rate without any loss in selectivity, which will be beneficial in industrial scale applications.
Keywords: Immunoglobulin G subclasses; Monolith; Separation; Protein A chromatography;

Long acting luteinizing hormone-releasing hormone (LHRH) antagonists designed to be protease-resistant were a series of novel decapeptides structurally similar to LHRH. In the present work, a high-throughput method based on a LC–MS/MS has been developed for the simultaneous determination of pharmacokinetics of five LHRH antagonists in rat via cassette dosing. The method was performed under selected reaction monitoring (SRM) in positive ion mode. The analytes were extracted from rat plasma by liquid–liquid extraction with acetonitrile. Chromatographic separation of the analytes was successfully achieved on a Hypersil Gold (100 mm × 2.1 mm, 3 μm) using a mobile phase composed of acetonitrile–water (30:70) containing 0.05% (v/v) formic acid. The result showed good linearity and selectivity were obtained for all antagonists. The limits of quantification of the five LHRH antagonists were from 5 to 10 ng/mL. The average extract recoveries in the rat plasma were all over 72%. The intra-day and inter-day precisions (R.S.D. %) were all within 10% and the accuracy was ranged from 92.54 to 109.05%. This method has been successfully applied to the pharmacokinetic studies of the five LHRH antagonists. The results indicated that the plasma drug concentrations versus time curves after intravenous injection of five antagonists via cassette dosing were all fitted to a two-compartment model. The pharmacokinetic parameters of five LHRH antagonists suggested that LY616 could be the more stable candidate drugs and optimized as the candidate drug for further study. Our studies enabled high-throughput rapid screening for pharmacokinetic assessment of new peptide candidates, and provided abundant information on the metabolic properties of these LHRH antagonists.
Keywords: LHRH antagonists; Peptide; Pharmacokinetics; Cassette dosing; LC–MS;

Vigabatrin in dried plasma spots: Validation of a novel LC–MS/MS method and application to clinical practice by Nađa Kostić; Yannis Dotsikas; Nebojša Jović; Galina Stevanović; Anđelija Malenović; Mirjana Medenica (102-108).
This paper presents a LC–MS/MS method for the determination of antiepileptic drug vigabatrin in dried plasma spots (DPS). Due to its zwitterionic chemical structure, a pre-column derivatization procedure was performed, aiming to yield enhanced ionization efficiency and improved chromatographic behaviour. Propyl chloroformate, in the presence of propanol, was selected as the best derivatization reagent, providing a strong signal along with reasonable run time. A relatively novel sample collection technique, DPS, was utilized, offering easy sample handling and analysis, using a sample in micro amount (∼5 μL). Derivatized vigabatrin and its internal standard, 4-aminocyclohexanecarboxylic acid, were extracted by liquid-liquid extraction (LLE) and determined in positive ion mode by applying two SRM transitions per analyte. A Zorbax Eclipse XDB-C8 column (150 × 4.6 mm, 5 μm particle size) maintained at 30 °C, was utilized with running mobile phase composed of acetonitrile: 0.15% formic acid (85:15, v/v). Flow rate was 550 μL/min and total run time 4.5 min. The assay exhibited excellent linearity over the concentration range of 0.500–50.0 μg/mL, which is suitable for the determination of vigabatrin level after per os administration in children and youths with epilepsy, who were on vigabatrin therapy, with or without co-medication. Specificity, accuracy, precision, recovery, matrix-effect and stability were also estimated and assessed within acceptance criteria.
Keywords: Vigabatrin; Chloroformates; Dried plasma spots; Mass spectrometry;

Analytical sample preparation strategies for the determination of antimalarial drugs in human whole blood, plasma and urine by Monica Escolà Casas; Martin Hansen; Kristine A. Krogh; Bjarne Styrishave; Erland Björklund (109-131).
Antimalarial drugs commonly referred to as antimalarials, include a variety of compounds with different physicochemical properties. There is a lack of information on antimalarial distribution in the body over time after administration, e.g. the drug concentrations in whole blood, plasma, and urine, which must be improved in order to advance curing the parasitic disease malaria. A key problem also lies in that pharmacokinetic studies not always are performed in patient groups that may benefit most of the treatment such as children, pregnancy and lower-weight ethnic populations. Here we review the available sample preparation strategies combined with liquid chromatographic (LC) analysis to determine antimalarials in whole blood, plasma and urine published over the last decade. Sample preparation can be done by protein precipitation, solid-phase extraction, liquid–liquid extraction or dilution. After LC separation, the preferred detection tool is tandem mass spectrometry (MS/MS) but other detection methods have been used e.g. UV, fluorescence and electrochemical detection. Major trends for sample preparation of the different groups of antimalarials for each matrix and its detection have been summarized. Finally, the main problems that the researchers have dealt with are highlighted. This information will aid analytical chemists in the development of novel methods for determining existing antimalarials and upcoming new drugs.
Keywords: Sample preparation; Antimalarials; Blood; Plasma; Urine;

Nobiliside A (Nob) is a new triterpenoid saponin separated from Holothuria noblilis. In this article, a liquid chromatography–electrospray ionization-tandem mass spectrometry method was established to quantify Nob, a hemolytic saponin, in rat blood and tissue homogenates. Standard curves were linear (r  = 0.9988–0.9995) over the range 50–5000 ng/mL in blood and 100–10000 ng/g in tissues. The lower limit of quantification (LLOQ) was 50 ng/mL for Nob. The novel method was rapid, accurate, highly sensitive and highly selective. Using this method, the pharmacokinetics and biodistribution of Nob liposome and Nob solution in Sprague-Dawley rats after a single intravenous dose of 1 mg/kg were then investigated. Nob was cleared slowly from circulation. There was no significant difference of the pharmacokinetic parameters in blood between Nob solution and Nob liposome. The highest AUC of Nob was observed in liver for the two groups, followed by spleen, lungs, kidney and heart. Compared with Nob solution, Nob liposome showed much higher AUC in liver and spleen and much lower AUC in kidney, heart and lung, which might be one important reason for the decreased toxicity of Nob.
Keywords: Nobiliside A; Liposome; LC/MS/MS; Pharmacokinetics; Bio-distribution;

A fast, accurate and precise ion chromatography method with pulsed amperometric detection was applied to evaluate a variety of parameters affecting the determination of total iodine in serum and urine of 81 subjects, including 56 obese and 25 healthy Polish children. The sample pretreatment methods were carried out in a closed system and with the assistance of microwaves. Both alkaline and acidic digestion procedures were developed and optimized to find the simplest combination of reagents and the appropriate parameters for digestion that would allow for the fastest, least time consuming and most cost-effective way of analysis. A good correlation between the certified and the measured concentrations was achieved. The best recoveries (96.8% for urine and 98.8% for serum samples) were achieved using 1 ml of 25% tetramethylammonium hydroxide solution within 6 min for 0.1 ml of serum/urine samples. Using 0.5 ml of 65% nitric acid solution the best recovery (95.3%) was obtained when 7 min of effective digestion time was used. Freeze–thaw stability and long-term stability were checked. After 24 weeks 14.7% loss of iodine in urine, and 10.9% in serum samples occurred. For urine samples, better correlation (R 2  = 0.9891) of various sample preparation procedures (alkaline digestion and application of OnGuard RP cartidges) was obtained. Significantly lower iodide content was found in samples taken from obese children. Serum iodine content in obese children was markedly variable in comparison with the healthy group, whereas the difference was less evident when urine samples were analyzed. The mean content in serum was 59.12 ± 8.86 μg/L, and in urine 98.26 ± 25.93 for obese children when samples were prepared by the use of optimized alkaline digestion reinforced by microwaves. In healthy children the mean content in serum was 82.58 ± 6.01 μg/L, and in urine 145.76 ± 31.44 μg/L.
Keywords: Iodine; Ion chromatography; Pulsed amperometric detection; Digestion procedures; Validation; Obesity;

Determination of irinotecan and its metabolite SN-38 in rabbit plasma and tumors using a validated method of tandem mass spectrometry coupled with liquid chromatography by Dae J. Park; Jun H. Won; A.R. Cho; Hye J. Yun; Jeong H. Heo; Tae H. Hwhang; Dae H. Lee; Woo M. Kim (147-152).
New tandem mass spectrometric method coupled with liquid chromatography (LC–MS/MS) has been developed to determine the total concentration of camptothecin derivatives (irinotecan and SN-38) regardless of inter-conversion phenomenon between carboxylate and lactone forms. At first, all sample solutions were acidified for 1 h in order to completely convert CPT derivatives into their lactone forms and then CPT derivatives were extracted with organic solution containing diethyl ether and ethyl acetate (2:1, v/v) just after alkalization in the range pH 8.0–8.5 in acid-treated solutions. Analytes were separated on a reverse phase C18 column (150 × 2.1 mm) and eluted isocratically with a mobile phase which consisted of acetonitrile-methanol-buffer (0.1% formic acid, 5 mM ammonium formate) (3:4:3, v/v). CPT derivatives were monitored by tandem mass spectrometry in electrospay-positive ionization and multiple reaction mode programmed to the following transitions (m/z): ‘587.6 → 167.2’ of CPT-11, ‘393.6 → 349.3’ of SN-38 and ‘349.4 → 305.2’ of CPT. The method was validated to have the proper linearity (r 2  > 0.99) over the range of 5–1000 ng/ml of CPT-11 and 1–250 ng/ml of SN-38 with good accuracy (89.8–114.3%) and precision (less than 10%). In all stability tests, concentration of CPT-11 and SN-38 had been left in the acceptable range of 88.8–110.7% when sample solutions were acidified before determination of CPT derivatives. Newly developed LC–MS/MS method was suitable for the determination of CPT derivatives of both rabbit plasma and tumor tissues in the pharmacokinetic study.
Keywords: Irinotecan; SN-38; Camptothecin; Rabbit; VX2 tumor; LC–MS/MS;