Precipitation Of Serum Proteins Research Articles

Analysis of 4-Hydroxyphenyllactic Acid and Other Diagnostically Important Metabolites of α-Amino Acids in Human Blood Serum Using a Validated and Sensitive Ultra-High-Pressure Liquid Chromatography-Tandem Mass Spectrometry Method.

The profile of and dynamic concentration changes in tyrosine, phenylalanine, and tryptophan metabolites in blood are of great interest since they could be considered potential biomarkers of different disorders. Some aromatic metabolites, such as 4-hydroxyphenyllactic, 4-hydroxyphenylacetic, phenyllactic, and 4-hydroxybenzoic acids have previously demonstrated their diagnostic significance in critically ill patients and patients with post-COVID-19 syndrome. In this study, a sensitive method, including serum protein precipitation with methanol and ultra-high-pressure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) detection, was developed and validated for six phenyl- and five indole-containing acids in human serum. The liquid-liquid extraction was also examined, but it demonstrated unsatisfactory results based on analyte recoveries and the matrix effect. However, the recoveries for all analytes reached 100% and matrix effects were not observed using protein precipitation. This made it possible to use deionized water as a blank matrix. The lower limits of quantitation (LLOQs) were from 0.02 to 0.25 μmol/L. The validated method was used for the analysis of serum samples of healthy volunteers (n = 48) to reveal the reference values of the target analytes. The concentrations of the most clinically significant metabolite 4-hydroxyphenyllactic acid, which were revealed using UPLC-MS/MS and a previously developed gas chromatography-mass spectrometry method, were completely comparable. The proposed UPLC-MS/MS protocol can be used in the routine clinical practice of medical centers.

A Rapid Method for Determination of Serum Methotrexate Using Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry and Its Application in Therapeutic Drug Monitoring.

Objectives Methotrexate (MTX) has anticancer therapeutic potential with multiple doses-related adverse effects and toxicities. Immunoassays for therapeutic monitoring of serum MTX have their own limitations. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered as the reference method; however, commercially availability of them is limited. We aimed to adapt/develop an in-house LC-MS/MS method for therapeutic monitoring of serum MTX. Materials and Methods Serum protein precipitation was performed using acetonitrile-water containing 250 μM solution of aminoacetophenone as internal standard (IS). Chromatographic separation was achieved on a C18 column with mobile phase of 0.1% solution of formic acid (solvent A) and acetonitrile (solvent B) at a flow rate of 0.4 mL/min. MS was performed under positive ion mode with mass transition for MTX and IS as m/z 455.1→308.1 and 136.2→94.1, respectively. The method was validated by following Bioanalytical Method Validation Guidance for Industry, 2018 and applied on leukemia patients' samples on MTX therapy. Results The correlation coefficient of eight serially diluted calibration standards of 0.09 to 12.5 μM was >0.99 and had linearity with > 95% precision and accuracy at analytical quality control levels. The lower limit of MTX quantification achieved was 0.09 μM with good intensity and sharp peak as compared with blank sample. The total run time of the assay was 5 minutes. The serum MTX levels obtained by this method in leukemia patients exhibited clinical correlation and an excellent agreement with commercial immunoassay used in parallel. Conclusion We were able to develop a rapid, sensitive, and cost-effective LC-MS/MS method suitable for therapeutic drug monitoring of MTX in routine clinical diagnostic laboratories.

Quantitative analysis of specific androgens in serum and urine samples from male, pre, and postmenopausal subjects using LC-MRM-MS

Androgens are endogenous hormones that play a crucial role in the paracrine and intracrine hormone system to perform and maintain vital physiological functions. Altered levels of androgens are implicated in many diseases such as sexual dysregulation, breast cancer, prostate cancer, and heart diseases etc. In this manuscript we describe a liquid chromatography-mass spectrometry (LC-MS) method using multiple reaction monitoring (MRM) for quantitatively measuring specific androgens such as dehydroepiandrosterone, testosterone, androsterone sulphate, androstenedione, and dihydrotestosterone in serum and urine samples. Serum acquired from nine different subjects (three pre-menopausal women, three postmenopausal women, and three healthy males) were used to evaluate the developed methods. In the sample preparation methods for serum either protein precipitation or liquid–liquid extraction (LLE) was used while the analysis of urinary androgens used LLE. The extracted androgens were quantitatively measured using LC-MRM-MS to which known amounts of stable isotope labeled standards were added. This manuscript also presents a LC-MRM-MS method mode for the analysis of oxime derivatized androgens potentially to enhance the sensitivity of the assay if required, from urine and venous-drawn serum samples.

Simultaneous Determination of Donepezil and its Two Metabolites in Human Serum by Liquid Chromatography Coupled With Mass Spectrometry

Alzheimer’s disease is a progressive neurodegenerative disease. Its incidence increases with aging of population and currently ranks among the most frequent dementia. Donepezil is indicated for the treatment of Alzheimer’s type dementia and acts as a centrally acting reversible acetyl cholinesterase (AChE) inhibitor. It is metabolized by CYP 450 isoenzymes 2D6 and 3A4 in the liver. The main metabolite, 6-O-desmethyldonepezil, has been reported to inhibit AChE to the same extent as donepezil. AGNP-TDM Expert Group recommended their routine therapeutic monitoring. We developed a new chromatographic method coupled with mass spectrometric detection to determination of donepezil, 5-O-desmethyldonepezil and 6-O-desmethyldonepezil in the human serum. Analysis was performed on a Waters UPLC H-class system (Waters, Milford MA, USA) in combination with XEVO TQD triple quadrupole (Micromass, Manchester, UK). Separation was made on BEH C18 column, using a mobile phase gradient (water: acetonitrile: ammonium acetate), time of analysis 7 min. Precipitation of serum proteins was performed with a precipitation reagent. The method was validated by the FDA rules in terms of repeatability, intermediate precision accuracy, matrix effect and stability. The coefficients of variation were found in the range from 2.77% to 12.04%, a recovery between 93.98 - 107.90% and the bias in the range from -10% to -3%. The presented method is applicable to routine therapeutic monitoring of donepezil and its two metabolites in the serum with simple sample preparation.

ANTI-HISTONE H1 IgGs POSSESS PROLIFERATIVE ACTIVITY TOWARDS HUMAN T-LEUKAEMIA CEM CELLS

Anti-histone H1 IgGs were purified from blood serum of systemic lupus erythematosus patients by precipitation of serum proteins with 50% ammonium sulfate followed by a sequential affinity chromatography on Protein G-Sepharose and histone H1-Sepharose columns. To avoid contamination with other proteins, anti-histone H1 IgGs were subjected to strongly acidic pH 2.0 during gel filtration through HPLC column. The effects of the anti-histone H1 IgGs on cell viability and cell cycle were tested by MTS-assay and flow cytometry, correspondingly. The cross-reactivity of the anti-histone H1 antibodies towards heterogenetic and cellular antigens was evaluated by Western-blot analysis. It was found that incubation of CEM cells with the HPLC-purified anti-histone H1 IgGs resulted in significant stimulation of cell growth by 46% after 48 h of incubation. These IgGs possess an antigenic poly-specificity to positively charged heterogenetic antigens and different cellular antigens. FITC-labeled and biotinylated anti-histone H1 IgGs are internalized by CEM cells and preferentially accumulated in the cytoplasm. The anti-histone H1 IgGs are shown to internalize human T-leukemia CEM and stimulate their proliferation. These IgGs are polyspecific toward cellular antigens.

Development of a liquid chromatography tandem mass spectrometry method for the simultaneous measurement of voriconazole, posaconazole and itraconazole.

Background Azole-based antifungals are the first-line therapy for some of the most common mycoses and are now also being used prophylactically to protect immunocompromised patients. However, due to variability in both their metabolism and bioavailability, therapeutic drug monitoring is essential to avoid toxicity but still gain maximum efficacy. Methods Following protein precipitation of serum with acetonitrile, 20 µL of extract was injected onto a 2.1 × 50 mm Waters Atlantis dC18 3 µm column. Detection was via a Waters Quattro Premier XE tandem mass spectrometer operating in ESI-positive mode. Multiple reaction monitoring (MRM) detected two product ions for each compound and one for each isotopically labelled internal standard. Ion suppression, linearity, stability, matrix effects, recovery, imprecision, lower limits of measuring interval and detection were all assessed. Results Optimal chromatographic separation was achieved using gradient elution over 8 minutes. Voriconazole, posaconazole and itraconazole eluted at 1.71, 2.73 and 3.41 min, respectively. The lower limits of measuring interval for all three compounds was 0.1 mg/L. The assay was linear to 10 mg/L for voriconazole (R2 = 0.995) and 5 mg/L for posaconazole (R2 = 0.990) and itraconazole (R2 = 0.991). The assay was both highly accurate and precise with % bias of voriconazole, posaconazole and itraconazole, respectively, when compared with previous NEQAS samples. The intra-assay precision (CV%) was 1.6%, 2.5% and 1.9% for voriconazole, posaconazole and itraconazole, respectively, across the linear range. Conclusion A simple and robust method has been validated for azole antifungal therapeutic drug monitoring. This new assay will result in a greatly improved sample turnaround time and will therefore vastly increase the clinical utility of azole antifungal drug monitoring.

Quantification of 1-(propan-2-ylamino)-4-propoxy-9H-thioxanthen-9-one (TX5), a newly synthetized P-glycoprotein inducer/activator, in biological samples: method development and validation.

A simple, rapid and economical method was developed and validated for the analysis and quantification of 1-(propan-2-ylamino)-4-propoxy-9H-thioxanthen-9-one (TX5), a P-glycoprotein inducer/activator, in biological samples, using reverse-phase high-performance liquid chromatography (HPLC). A C18 column and a mobile phase composed of methanol-water (90/10, v/v) with 1% (v/v) triethylamine, at a flow rate of 1 mL/min, were used for chromatographic separation. TX5 standards (0.5-150 μm) were prepared in human serum. Methanol was used for TX5 extraction and serum protein precipitation. After filtration, samples were injected into the HPLC apparatus and TX5 was quantified by a conventional UV detector at 255 nm. The TX5 retention time was 13 min in this isocratic system. The method was validated according to ICH guidelines for specificity/selectivity, linearity, accuracy, precision, limits of detection and quantification (LOD and LOQ) and recovery. The method was proved to be selective, as there were no interferences of endogenous compounds with the same retention time of TX5. Also, the developed method was linear (r2 ≥ 0.99) for TX5 concentrations between 0.5 and 150 μm and the LOD and LOQ were 0.08 and 0.23 μm, respectively. The results indicated that the reported method could meet the requirements for TX5 analysis in the trace amounts expected to be present in biological samples.

Experimental and Computational Thermal Modeling of In Vitro Pin-on-Disk Tests of Ultra High Molecular Weight Polyethylene

Frictional heating occurring during pin-on-flat tribotesting of ultrahigh molecular weight polyethylene (UHMWPE) pins was measured and modeled. A full factorial experiment was conducted to determine if testing parameters can produce sufficient frictional heat to alter tribological properties of the bovine serum used as lubricant in the system. Temperature of the surrounding bovine serum was monitored during tribotests using varying pin sizes and sliding speeds to determine typical temperature rises due to frictional heating. This work examined two sliding speeds (40 mm/s and 80 mm/s) and two pin diameters (6.35 mm and 9.5 mm) at a single static load. Gravimetric analysis for wear determination and coefficient of friction measurement were performed for each test. Results showed that frictional heating increased the bulk temperature of the surrounding serum and correlated to sliding speed and average coefficient of friction. No correlation was seen at this temperature range between serum temperature rise and wear rate, providing evidence that the tested parameters are acceptable for tribotesting of UHMWPE. A computational model was developed to predict bulk serum temperature increase. This model closely predicted the temperature increase to within 2 °C, which is sufficient accuracy for identifying if bovine serum protein precipitation is likely during tribotesting. This work serves as an initial estimate and prediction for appropriate testing parameters based on lubricant responses to frictional heating.

Comparison of different serum sample extraction methods and their suitability for mass spectrometry analysis

Mass spectrometry has been widely used, particularly in pharmacokinetic investigations and for therapeutic drug monitoring purposes. Like any other analytical method some difficulties exist in employing mass spectrometry, mainly when it is used to test biological samples, such as to detect drug candidates in mammalian serum, which is rich in proteins, lipids and other contents that may interfere with the investigational drug. The complexity of the serum proteome presents challenges for efficient sample preparation and adequate sensitivity for mass spectrometry analysis of drugs. Enrichment procedures prior to the drug analysis are often needed and as a result, the study of serum or plasma components usually demands either methods of purification or depletion of one or more. Selection of the best combination of sample introduction method is a crucial determinant of the sensitivity and accuracy of mass spectrometry. The aim of this study was to determine the highest serum protein precipitation activity of five commonly used sample preparation methods and test their suitability for mass spectrometry. We spiked three small molecules into rabbit serum and applied different protein precipitation methods to determine their precipitation activity and applicability as a mass spectrometry introductory tool.

Sensitive routine liquid chromatography–tandem mass spectrometry method for serum estradiol and estrone without derivatization

The need for a routinely applicable assay to measure low estradiol levels in adult men, postmenopausal women, and young adolescents was recently discussed in an Endocrine Society position statement. Our aim was to develop a sensitive liquid chromatography-tandem mass spectrometry method for estradiol and estrone in human serum without the need for derivatization or extended extraction protocols. After protein precipitation of serum with a mixture of methanol/acetonitrile (85/15) (v/v) containing isotopic internal standards (17β-estradiol-16,16,17-d 3 and estrone-2,3,4-(13)C), we quantified estradiol and estrone by two-dimensional liquid chromatography-tandem mass spectrometry with electrospray ionization in the negative mode monitoring 5 × 271.20→145.00 (17β-estradiol) and 269.20→145.00 (estrone). Sensitivity was increased by using fluoride and summation of 5 identical transitions for estradiol. Our method was analytically validated, compared against direct immunoassays using serum of 25 adult men, and clinically tested by measuring samples of 3 men at baseline and after chemical castration, 30 postmenopausal women and 15 patients receiving aromatase inhibitors. Total imprecision was below 20% for the low quality controls. Limit of quantification was 1.3 ng/L (4.8 pmol/L) for estradiol and 1.2 ng/L (4.4 pmol/L) for estrone. Estradiol in Certified Reference Material BCR-576 was within specified uncertainty limits. No significant ion suppression or interference was observed. Our method showed modest correlation with direct immunoassay for estradiol (r(2) = 0.64) but no correlation for estrone (r(2) = 0.12). Patient sample results were within expected ranges. In conclusion, we developed a routinely applicable liquid chromatography-tandem mass spectrometry method for estradiol and estrone measurement which is sensitive enough for use in men, postmenopausal women, and young adolescents.

Fully validated method for rapid and simultaneous measurement of six antiepileptic drugs in serum and plasma using ultra-performance liquid chromatography–electrospray ionization tandem mass spectrometry

Therapeutic drug monitoring (TDM) may be very useful in the clinical management of antiepileptic drug therapy for multiple reasons, such as individual variability, metabolism, genetic factors or drug–drug or drug–food interactions. In addition, TDM is helpful to study the variation in pharmacokinetics that occurs between individuals. Here, we describe a rapid assay using ultra-performance liquid chromatography–electrospray ionization tandem mass spectrometry to measure the antiepileptic drugs lacosamide, lamotrigine, levetiracetam, primidone, topiramate, and zonisamide. After the addition of internal standards (ISs) and protein precipitation of serum or plasma, 1μl of sample was separated on a 2.1×50mm reverse phase column (Waters, Acquity UPLC BEH Phenyl, 1.7μm). Analytes were then ionized and detected by electrospray ionization mass spectrometry with multiple reaction monitoring. Runtime was 2.5min per injection. Matrix effects were investigated by systematical ion suppression and in-source fragmentation experiments. The calibration curves of the 6 antiepileptic drugs were linear over the working range between 0.05 and 50mg/L (r>0.99). The limit of detection (LOD) was <0.05mg/L, whereas the limit of quantification (LLOQ) was 0.10mg/L of all drugs measured in the assay. The intraassay and interassay coefficients of variation for all compounds were <15% for very low concentration (0.1mg/L) and <8% in the clinically relevant concentration range (>1.0mg/L). Mean recoveries were between 87.8 and 98.6% for all drugs. There were no significant ion suppressions detected at the elution times of the analytes. The mean differences between serum and heparinized plasma values were less than 6% for the 6 antiepileptic drugs. All drugs were stable in serum at −20°C, 4°C, and even at RT for at least 1 month. In summary, a specific and sensitive stable isotope dilution UPLC-MS/MS method was developed and validated for routine clinical monitoring of lacosamide, lamotrigine, levetiracetam, primidone, topiramate, and zonisamide.

Simple HPLC method for cefazolin determination in human serum – validation and stability testing

The paper presents an HPLC method for cefazolin determination in human serum. The preparation step was based on serum protein precipitation with acetonitrile followed by supernatant evaporation and sample reconstitution in water before injection. The separation of cefazolin and internal standard cefamandole was performed at ambient temperature under isocratic conditions on LiChrosorb RP8-5 column (250mm×4.6mm) using the mixture: CH3CN:H2O:0.5M KH2PO4 (100:894:6, v/v) as a mobile phase with a flow rate of 1.5mL/min. UV detection was performed at 272nm with LLOQ of 0.2μg/mL. The precision was satisfactory in the whole range tested with RSD of 2.3–12.5% (accuracy: from −2.3% to +3.6%) and of 1.7–7.1% (accuracy: from −3.5% to +1.1%) for intra- and inter-assay, respectively. The method stability was confirmed in a series of experiments including: freeze–thaw and short- and long-term stability testing. Finally, the procedure described was found resistant to potential human errors.

Liquid chromatography–tandem mass spectrometry method for determination of five antidepressants and four atypical antipsychotics and their main metabolites in human serum

The rapid and simple ultra performance liquid chromatography–tandem mass spectrometry method was developed and validated for simultaneous determination parent drugs: sertraline, fluoxetine, citalopram, paroxetine, venlafaxine, clozapine, olanzapine, quetiapine, risperidone, and their active and nonactive metabolites N-desmethylsertraline, norfluoxetine, desmethylcitalopram, didemethylcitalopram, N-desmethylvenlafaxine, O-desmethylvenlafaxine, N-desmethylclozapine, N-desmethylolanzapine, 2-hydroxyolanzapine and 9-hydroxyrisperidone in human serum. Precipitation of serum proteins was performed with a precipitation reagent consisting of 0.05% solution of ZnSO4·7H2O in acetonitrile/methanol (40:60, v/v). Alprenolol was used as an internal standard. Chromatographic separation was carried out on a BEH C18 column using gradient elution mobile phase A (2mmol/L ammonium acetate, 0.1% formic acid in 5% acetonitrile, v/v/v) and B (2mmol/L ammonium acetate, 0.1% formic acid in 95% acetonitrile, v/v/v). Electrospray in positive mode was used for ionization. Detection was performed on a triple–quadrupole tandem mass spectrometer by multiple reaction monitoring. Analysis time was 5min. Drugs were separated into three groups with low, medium and high levels. Correlation coefficients of calibration curves were in the range 0.995–1.000. Coefficients of variation were 4.2–9.5% for intra-assay and 3.0–11.9% for inter-assay. Recoveries were 87.1–110% for intra-assay and 88.1–108.2% for inter-assay. The method was fully validated and can be successfully applied for routine analyses.

Rapid extraction, identification and quantification of oral hypoglycaemic drugs in serum and hair using LC–MS/MS

A sensitive and accurate LC–MS/MS method for the identification and quantification of 5 oral anti-diabetics (glipizide, glibenclamide, gliclazide, gliquidone and metformin) in serum and hair was developed using glibornuride as the internal standard. We have developed a rapid and robust extraction procedure by using acetonitrile for serum protein precipitation and methanol for the extraction of anti-diabetics from hair. Anti-diabetics (ADs) were separated by UPLC over a C18 column and detection was performed on a Waters Xevo TQ MS mass spectrometer in positive ionization mode using electrospray ionization. Each AD was identified by three specific ion transitions in multiple reaction monitoring (MRM) mode. The method was validated according to international guidelines. For all compounds the variation coefficient (CV) was <20%, and accuracies ranged from 85 to 115% in serum and hair. The limits of detection (LODs) were <1.5ng/mL for all ADs in serum and <3.59pg/mg in hair. Recoveries varied from 56.41% (gliclazide) to 67.58% (glipizide) in serum and from 68% (gliclazide) to 91.2% (metformin) in hair. The method was successfully applied to quantify ADs in serum of 33 patients and in hair of 15 patients.

Trisodium citrate induced protein precipitation in haemodialysis catheters might cause pulmonary embolism

The locking anticoagulant plays a decisive role in the patency of central venous catheters (CVCs) used for haemodialysis. During injection, the hydraulic effects inevitably cause lock solution to spill into the systemic circulation. Density differences between whole blood (WB) and the lock solution cause further gravity-induced seepage of lock solution. This is followed by an influx of WB into the catheter, also described for trisodium citrate, which is a common agent for serum protein precipitation. Embolic complications from haemodialysis catheters locked with hypertonic trisodium citrate have been reported. We aimed to investigate protein precipitation in trisodium citrate locked catheters as a possible cause of pulmonary embolisms. In vitro, WB and trisodium citrate (concentrations ranging from 4.7 to 46.7%) mixtures in a ratio of 1:4 were used to assess protein precipitation. Additionally, WB/trisodium citrate mixture was pumped through a 20-μm mesh filter, simulating pulmonary vessels, and filtrate pressure was measured. In vivo, listed filling volumes of haemodialysis catheters locked with trisodium citrate 4% (n=10), 10% (n=10), 20% (n=10) or 46.7% (n=10) were aspirated and then analysed for protein precipitation. In vitro, protein precipitation capable of causing filter occlusion was observed in test solutions containing trisodium citrate above a concentration of 12%. In vivo, protein precipitation was detected in all samples from the CVCs filled with trisodium citrate 46.7% (n=10) and 20% (n=10). In contrast, there were no signs of precipitation in samples from the catheters filled with trisodium citrate 4% (n=10) or 10% (n=10). Our in vitro results demonstrate that protein precipitates inside haemodialysis catheters when trisodium citrate is used above the concentrations of 12%. Precipitated protein may have contributed to the pathophysiology of reported embolisms from haemodialysis catheters filled with hypertonic trisodium citrate. Based on our findings, we suggest that trisodium citrate lock solution up to the concentration of 10% can be used safely.

Undetectable phenytoin serum levels by an automated particle-enhanced turbidimetric inhibition immunoassay in a patient with monoclonal IgMλ

IntroductionPhenytoin is a drug used for the treatment of different types of seizures. Its variable pharmacokinetics, mainly a consequence of variable bioavailability, saturable protein binding and saturable hepatic metabolism, predisposes the drug to therapeutic drug monitoring. Several methods to analyze the drug in serum exist with immunoassays being the method of choice for routine measurements. Immunoassays are specific and sensitive, but cross-reactivity, possibly leading to erroneous serum levels, is a concern. We report a patient with falsely undetectable phenytoin serum levels. Case reportThis 73-year old woman was treated with intravenous phenytoin due to epilepsia partialis continua in the context of a bilateral cerebrovascular insult. Anamnestically, a chronic lymphatic leukemia was known. In this patient, serum phenytoin levels became only detectable by the particle enhanced turbidimetric inhibition immunoassay used after precipitation of serum proteins. Protein electrophoresis revealed a monoclonal immunoglobulin, identified as IgMλ. With other methods such as HPLC and fluorescence depolarization immunoassay, phenytoin was detectable. ConclusionWe propose interference between the monoclonal IgMλ and/or other serum proteins and the particle-enhanced turbidimetric inhibition immunoassay, rendering phenytoin falsely undetectable in samples of this patient. In such patients, alternative methods such as HPLC should be used to prevent dosage errors.

Sensitive and convenient method for the quantification of clonidine in serum of pediatric patients using liquid chromatography/tandem mass spectrometry

An improved and easy to use liquid chromatography/tandem mass spectrometric (LC/MS/MS) method in human serum was developed for the quantification of clonidine (CLD), an alpha 2-/alpha 1-adrenoceptor agonist, used for analgo-sedation and the therapy of opioid withdrawal in pediatric patients. Sample preparation consisted of precipitation of serum proteins by adding acetonitrile and centrifugation of the sample subsequently. [ 2H 4]Clonidine (CLD4) served as internal standard. Chromatographic separation of the supernatant was achieved using a 100 mm × 3 mm, 5 μm Thermo Electron BetaBasic C4 column with isocratic flow and elution consisting of 0.1% formic acid/acetonitrile (85/15, v/v) and a flow-rate of 350 μl/min resulting in a column pressure of 280–420 kPa. LC/MS/MS detection was performed by using a triple-stage quadrupole mass spectrometer (TSQ Quantum, Thermo Electron) working in selected reaction monitoring mode with positive electrospray ionization. The analyte was quantified in a single run within 5 min. Linearity was demonstrated over the expected concentration range 0.15–50 μg/l CLD. The lower limit of quantification (LLOQ) and the limit of detection were 0.1 μg/l and 0.01 μg/l, respectively. None of the drugs used concomitantly during analgesic therapy interfered in the assay in vitro. Intra-day precision expressed as RSD was 9.6% or less for CLD, while inter-day result was 10.0% or less for CLD. Intra-day and inter-day accuracy was within ±4.9% and ±1.8%, respectively. The method was validated according to the international guidelines of the International Conference on Harmonisation (ICH) and the US Food and Drugs Administration (FDA). The described method is suitable to analyse serum samples with very small volumes and sets the stage for pharmacokinetic studies in pediatric studies.

Pilot Study on the Perfluorooctanesulfonate and Perfluorooctanoic Acid Exposure of the General Population

MAA4-PD-05 Introduction: Perfluoro-octanesulfonate (PFOS) and perfluoro-octanoic acid (PFOA) are raw materials or used as manufacturing aids for some perfluorinated chemicals and can be released of those by biotic and/or metabolic decomposition. Because of their widespread use, persistence, and bioaccumulative properties, they are taken up by the general population from different sources. In animal studies, both substances provoked various types of cancer and showed developmental toxic potential besides other adverse health effects. To receive an impression about the internal exposure of the general population against PFOS and PFOA, we determined these substances in 105 plasma samples. Methods: A total of 105 nonsmoking subjects (54 male, 51 female, 5–84 years of age; white) without a known occupational exposure to PFOS or PFOA took part in the study. Seven equal age groups were formed to guarantee a uniform age distribution and to find out if plasma concentrations are possibly influenced by the age of the subjects. The age groups consisted of one-half male and female subjects, respectively. An analytical method based on serum protein precipitation followed by HPLC with MS/MS-detection was used. As an internal standard, we used isotopically labelled 1,2-13C-PFOA. Results: We found PFOS and PFOA in every plasma sample analyzed. PFOS plasma concentrations ranged from 6.2 μg/L to 130.7 μg/L (median, 22.3 μg/L) and were continuously higher than those of PFOA, which we determined in concentrations from 1.7 μg/L to 39.3 μg/L (median, 6.8 μg/L). No significant age trend of plasma concentrations could be revealed. Considering the gender of the subjects, we found a significant difference between men and women with men having both higher PFOS and PFOA levels (P < 0.05). A strong correlation was found between PFOS and PFOA (r = 0.82). Discussion and Conclusions: We could show a ubiquitous exposure of the general population against PFOS and PFOA. Furthermore, we demonstrated the influence of the gender on plasma concentrations. An age trend of plasma concentrations was not observable. Therefore, we think that the existing information about the long half-lives of PFOS and PFOA should be confirmed in further studies. The strong correlation between PFOS and PFOA is most probably due to common sources of both substances, so future research activities should also aim on the clarification of those.

Determination of Perfluorooctanesulfonate and Perfluorooctanoic Acid in Maternal and Cord Blood Samples

SAB2-O-06 Introduction: Perfluoro-octanesulfonate (PFOS) and Perfluoro-octanoic acid (PFOA) are raw materials or used as manufacturing aids for some perfluorinated chemicals and can be released of those by biotic and/or metabolic decomposition. Because of their ubiquitous occurrence, persistence, and bioaccumulative properties, they can be found in plasma and serum samples of the general population all over the world. In animal studies, PFOS and PFOA provoked various types of cancer and showed developmental toxic potential besides other adverse health effects. On the basis of the comparison of plasma sample pairs from mothers and their infants’ cord blood, we wanted to show if neonates are exposed to PFOS and PFOA in earliest state of development. Methods: We determined PFOS and PFOA in 11 plasma samples of mothers and in 11 plasma samples from the cord blood of their infants. An analytical method based on serum protein precipitation followed by HPLC with MS/MS-detection was used. As internal standards, we used isotopically labelled 1,2,3,4-,13C-PFOS and 1,2-13C-PFOA. Results: We found PFOS and PFOA in every plasma sample analyzed. In maternal plasma samples PFOS concentrations were consistently higher compared with those of the related cord plasma samples (median, 12.7 μg/L vs. 7.2 μg/L). In the case of PFOA, we observed only minor differences between PFOA concentrations within the analyzed sample pairs (median, 2.4 μg/L vs. 3.1 μg/L for maternal and cord plasma samples, respectively). Discussion and Conclusions: For both substances, a crossing of the placental barrier could be shown. For PFOS, we observed a decrease from maternal to cord plasma concentrations by a factor of 0.41 to 0.80. However, PFOA obviously crosses the placental barrier unhindered. These findings show that neonates are exposed to PFOS and PFOA via their mothers’ blood. Given the current situation that only little is known about the consequences of PFOS and PFOA exposure in the early state of development of humans and the fact that in animal studies both substances showed developmental toxic effects future research regarding human health effects is indispensable.

Analysis of creatinine in mouse and rat serum by ion exchange high performance liquid chromatography for in vivo studies of renal function

An ion exchange high performance liquid chromatography method was developed for determining creatinine levels in both mouse and rat serum samples. Separation of creatinine from other serum components was achieved in 10 min using a 100 × 4.1-mm, 10 μm strong cation exchange column following acetonitrile precipitation of serum proteins. Incorporation of a guard cartridge placed in-line prior to the analytical column was employed to prevent interference from compounds used in renal disease animal trials. Creatinine levels in normal and diseased animals were accurately determined in the 0.01–10 mg/dL range, and average recovery of the method was approximately 85% for both mouse and rat serum. Addition of 0.5–1.0% acetic acid to the acetonitrile used for protein precipitation significantly improved creatinine recovery to above 97% in mouse serum. The method was used for routine preclinical diagnosis of rat and mouse model renal function, and for the evaluation of renal disease treatment efficacy.