Sample Pretreatment Procedure Research Articles

In-syringe dispersive solid phase extraction: a novel format for electrospun fiber based microextraction.

A novel in-syringe dispersive solid phase extraction (dSPE) system using electrospun silica fibers as adsorbents has been developed in the current work. A few milligrams of electrospun silica fibers were incubated in sample solution in the barrel of a syringe for microextraction assisted by vortex. Due to the benefit of dispersion and the high mass transfer rate of the sub-microscale electrospun silica fibers, the extraction equilibrium was achieved in a very short time (less than 1 min). Moreover, thanks to the long fibrous properties of electrospun fibers, the separation of the adsorbent from sample solution was easily achieved by pushing out the sample solution which therefore simplified the sample pretreatment procedure. Besides, the analytical throughput was largely increased by using a multi-syringe plate to perform the extraction experiment. The performance of the in-syringe dSPE device was evaluated by extraction of endogenous cytokinins from plant tissue samples based on the hydrophilic interaction. Six endogenous cytokinins in 20 mg of Oryza sativa L. (O. sativa) leaves were successfully determined under optimized conditions using in-syringe dSPE combined with liquid chromatography-mass spectrometry analysis. The results demonstrated that the in-syringe dSPE method was a rapid and high-throughput strategy for the extraction of target compounds, which has great potential in microscale sample pretreatment using electrospun fibers.

Determination of clenbuterol from pork samples using surface molecularly imprinted polymers as the selective sorbents for microextraction in packed syringe

In this study, a selective sample pretreatment procedure combing surface molecularly imprinted polymers and microextraction in packed syringe (SMIPs-MEPS) was developed for the analysis of clenbuterol (CLB) from pork samples. SMIPs for CLB were synthesized on silica gel particles through a sol–gel process. A series of characterization and adsorption experiments revealed that the SMIPs exhibited porous structures, good thermal stability, high adsorption capacity and a fast mass transfer rate. The obtained SMIPs were employed as selective sorbents of SMIPs-MEPS for extraction of CLB from pork samples. Several parameters affecting the extraction efficiency were investigated, including the pH of sample solution, number of draw-eject cycles, volume of sample, type and volume of washing solution, and the type and volume of elution solution. Under the optimized conditions, a simple and rapid method for the determination of CLB from pork samples was established by coupling with high performance liquid chromatography (HPLC). The whole pretreatment process was rapid and it can be accomplished with 2min. The limit of quantitation and the limit of detection for CLB were 0.02 and 0.009μgkg−1, respectively. The average recoveries of CLB at three spiked levels ranged from 86.5% to 91.2% with the relative standard deviations (RSD) ≤6.3%.

자동연속흐름-흡광광도법과 DNPH-LC법에 의한 먹는물 중 포름알데히드 정량분석 비교

Due to the stringent drinking water quality, formaldehyde will be included in Korean drinking water standard from year 2014. However, its standard analytical method has not yet been established. This study compares two analytical methods, DNPH-LC and ACFAS with respect to their analysis principles, Method Detection Limit (MDL), Limit Of Quantitation(LOQ), precision, accuracy, reproducibility, convenience, number of samples analyzed per hour and analysis cost. These methods measure absorption intensity at 360 nm by using HPLC after DNPH-derivatization (DNPH-LC) and at 410 nm by using Automated Continuous Flow Absorption Spectrophotometer (ACFAS), respectively. Reproducibility was tested by repeating the analysis 7 times using a standard solution for each method. For DNPH-LC method, MDL was 0.5 μg/L, LOQ was 1.58 μg/L with standard deviation of 0.16 μg/L. For ACFAS method, they were 0.27 μg/L, 0.85 μg/L with standard deviation of 0.09 μg/L, respectively. Both methods satisfied the requirement set by the Korean drinking water quality standard. Complexity of sample pretreatment procedure for DNPH-LC method may cause large error and, consequently, the analytical result will depend on the level of skill of analyst. In contrast, ACFAS method which used only one reagent equipped with an automated injection device showed little analytical error. It costs about $5.00 and $1.00 for one sample to analyze by the DNPH-LC method and the ACFAS method, respectively. Compared to the DNPH-LC method, ACFAS method provided more reliable analytical results. In terms of convenience, easiness and analytical cost, ACFAS method was demonstrated to be superior to the DNPH-LC method. The results of this study suggested that the ACFAS method could be adapted as a proper method for determining formaldehyde content in drinking water.

Simultaneous determination of β-lactam antibiotics and β-lactamase inhibitors in bovine milk by ultra performance liquid chromatography-tandem mass spectrometry

An ultra performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method has been developed for the simultaneous determination of four β-lactam antibiotics (amoxicillin, ampicillin, cefotaxime, and cefoperazone) and two β-lactamase inhibitors (tazobactam, sulbactam) in bovine milk. The analytes were extracted with water from bovine milk and purified with Oasis HLB solid phase extraction (SPE) cartridges. The analytes were determined in less than 3min by UPLC-MS/MS in positive and negative electrospray ionization (ESI) modes, separately. The method was linear over the range of 1-100μg/L for tazobactam, sulbactam, ampicillin, and cefoperazone, and 2-100μg/L for amoxicillin and cefotaxime. The recoveries for all six analytes in bovine milk ranged from 82.5 to 98.3%. The limits of detection and the limits of quantitation were 0.1-0.2μg/L and 0.3-0.5μg/L, respectively. The intra- and inter-day precisions were less than 6% for each compound.

Development of multiresidue DLLME and QuEChERS based LC–MS/MS method for determination of selected neonicotinoid insecticides in honey liqueur

Honey liqueur is an alcoholic drink derived from honey and strong fruit brandy of suitable type, traditionally made in Serbia and other Balkan countries. Although European Union has regulated the levels of neonicotinoid insecticides in honey and pollen, there is an increased risk of the presence of these compounds in traditional products made from honey. The objective of this study was to develop an optimized LC–MS/MS analytical method with dispersive liquid–liquid microextraction (DLLME) and QuEChERS sample preparation procedures for analysis of seven neonicotinoids (dinotefuran, nitenpyram, thiamethoxam, clothianidin, imidacloprid, acetamiprid and thiacloprid) in honey liqueur. The LC–MS/MS conditions were optimized to unequivocally provide good chromatographic separation, selectivity and specificity of developed method. The method was validated to fulfill the requirements of SANCO/12495/2011 for both sample pretreatment procedures providing results for accuracy (R, 69.2–113.4% for DLLME; 71.8–94.9% for QuEChERS), precision (RSD expressed in terms of repeatability (3.21–10.20% for DLLME; 4.19–12.81% for QuEChERS) and within-laboratory reproducibility (9.11–16.63% for DLLME; 11.32–16.40% for QuEChERS)), limits of detection (LOD, 0.5–1.5μgL−1 for DLLME; 1.0–2.5μgL−1 for QuEChERS) and quantification (LOQ, 1.0–5.0μgL−1 for DLLME; 2.5–10.0μgL−1 for QuEChERS). Matrix effects were compensated by the use of matrix-matched calibration. Analysis of real honey liqueur samples obtained from local markets showed the presence of clothianidin or thiacloprid in four of the analyzed samples, therefore implicating the necessity of ongoing control of this type of traditional product.

HILIC UHPLC–MS/MS for Fast and Sensitive Bioanalysis: Accounting for Matrix Effects in Method Development

Matrix effects are considered to be a main obstacle of quantitative bioanalytical LC-MS/MS methods. Therefore it is often required to minimize them in order to increase method reliability. HILIC has been referenced as one of possible approaches. However, there is a lack of experimental evidence in scientific literature so far. Matrix effects were evaluated using spiked serum samples after SPE and protein precipitation prior to UHPLC-ESI-MS/MS. Chromatography was performed in both HILIC and reversed-phase mode. The influence of the matrix effects on the signal response was assessed using a set of 34 compounds of pharmaceutical interest and post-extraction addition approach. The advantages and drawbacks of the HILIC and reversed-phase chromatographic modes were compared and discussed in detail. HILIC demonstrated the potential to reduce the occurrence of matrix effects when a more thorough sample pretreatment procedure such as SPE was applied.

Sample pretreatment of brain tissues and cerebrospinal fluid for NMR investigations

Samples of rat homogenated brain cortex and cerebrospinal fluid prepared by various protocols were investigated by high-resolution NMR. The stability of the major metabolites, biomarkers of neurode-generative diseases, was examined depending on the procedure of sample pretreatment. As shown, the optimal conditions of the sample pretreatment of brain tissues to NMR analysis involve mechanical homogenization followed by the addition of deuterated water and for prolong experiments, an extraction with a methanol-chloroform mixture (2: 1). Samples of cerebrospinal fluid for NMR study should be prepared in buffer media based on deuterated water to improve the stability of the preparation and prevent the drift of metabolite signals in the spectra of different samples during the experimentation. The chosen optimal conditions of sample pretreatment of brain tissue and cerebrospinal fluid can be used in future to study changes in the composition and concentration of metabolites in neurodegenerative diseases by virtue of NMR. The proposed methods allow more accurate the diagnosis of pathologies of the brain function at early stages.

Quantitative analysis of thymosin β4 in whole saliva by capillary electrophoresis–mass spectrometry using multiple ions monitoring (CE‐MIM‐MS)

Thymosin β4 (Tβ4) is a peptide present in almost any tissue and in extracellular media in mammals, having multiple amazing functions as wound healing, stimulation of angiogenesis, and suppression of inflammation. This study describes its determination in saliva through CE-MS using multiple ions monitoring scan mode by isolating the four most intense multicharged ions present in the MS spectra of the peptide. This scan modality, by reducing the baseline noise and interferences, increases the sensitivity and specificity in biological matrices. The CE-MS separation was optimized by studying different parameters influencing CE analysis, sample injection, and MS ionization, that is, the nebulizer gas flow, the sheath liquid, and BGE composition. The proposed technique can unambiguously identify in short time Tβ4 in saliva after a very fast and reduced sample pretreatment procedure. The method was validated for quantitation showing linearity of the response in the range 0.25 (lower limit of quantification) to 4 μM (average R2 0.996 ± 0.005) and intra- and interassay precision and accuracy at three different concentrations with RSD values in the range of 7–16%. It was successfully applied to the analysis of Tβ4 in whole saliva showing a variable peptide content from individual to individual (in the range of 0.3–1.4 μM) and in different days from the same individual. CE-MS in multiple ions monitoring scan mode provides a fast, selective, and economic method requiring only very few microliters of sample.

Trace determination of lenalidomide in plasma by non-extractive HPLC procedures with fluorescence detection after pre-column derivatization with fluorescamine

BackgroundLenalidomide (LND) is a new potent drug used for treatment of multiple myeloma. For its pharmacokinetic studies and therapeutic monitoring, a proper analytical method was required.ResultsIn this study, a non extractive and simple pre-column derivatization procedures have been proposed, for the for trace determination of lenalidomide (LND) in human plasma by HPLC with fluorescence detection. Plasma samples were treated with acetonitrile for protein precipitation then treated with copper acetate to form stable complexes with the biogenic amines and mask their interference with the derivatization reaction of LND. Treated plasma samples containing LND was derivatized with fluorescamine (FLC) in aqueous media at ambient temperature. Separation of the derivatized LND was performed on Hypersil BDS C18 column (250 × 4.6 mm, 5 μm particle size) using a mobile phase consisting of phosphate buffer (pH 4):methanol: tetrahydrofuran (70:10:20, v/v) at a flow rate of 1.0 mL/min. The derivatized samples were monitored at an emission wavelength of 495 nm after excitation at a wavelength of 382 nm. Under the optimum chromatographic conditions, a linear relationship with good correlation coefficient (r = 0.9997, n = 9) was found between the peak area and LND concentrations in the range of 2–100 ng/mL. The limits of detection and quantitation were 0.8 and 2.30 ng/mL, respectively. The intra- and inter-assay precisions were satisfactory and the accuracy of the method was proved. The recovery of LND from the spiked human plasma was 99.30 ± 2.88.ConclusionsThe proposed method had high throughput as the analysis involved simple sample pre-treatment procedure and a relatively short run-time (< 15 min). The results demonstrated that the method would have a great value when it is applied in the therapeutic monitoring and pharmacokinetic studies for LND.

Highly sensitive and selective organophosphate screening in twelve commodities of fruits, vegetables and herbal medicines by dispersive liquid–liquid microextraction

The article describes a simple sample pretreatment procedure for the analysis of ten organophosphorus pesticides using dispersive liquid–liquid microextraction (DLLME) followed by gas chromatography–mass spectrometry (GC–MS) in three distinctively different types of matrices: fresh fruits, fresh vegetables and dried herbs. The method was carefully developed, focusing on the chemistry of various dispersive solvents, to achieve simultaneous, comprehensive extraction and preconcentration in a great span of selected matrices. According to matrix-matched validation study, the set of optimized DLLME conditions has been proven robust to determine target OPPs within a wide linear range from 0.1 to 1000μgL−1. With limited usage of organic extractants, remarkable enrichment factors up to 100-fold were obtained, enabling ultra-trace pesticide quantification down to sub-ppt levels at 0.12–4.92ngkg−1. Practical application of the method was illustrated by quantitative recovery (70–119%) and good precision (2.6–10% R.S.D.) in a representative range of three fruits and four vegetable commodities featured by the CODEX Alimentarius classification as well as their unique matrix compositions. A careful selection of dried herbs was further classified based on their morphological structures to validate analytical ruggedness of the method. Compared with existing methods for food analysis vis-à-vis OPPs, the present method is superior in terms of high sample throughput, minimal solvent consumption, and small sample size requirement. An additional, significant aspect of this universal DLLME method is that it models sample pretreatment methods with wide coverage of analytical matrices that are more effective, more comprehensive, and more flexible than those currently being used.

Preparation and validation of monoclonal antibody-based indirect competitive ELISA for detecting testosterone levels

This paper presents the generation of monoclonal antibodies (mAbs) against testosterone (TES) through cell fusion technology, and the development of a mAb-based indirect competitive enzyme-linked immunosorbent assay (icELISA) method to detect TES residue in bovine edible tissues. Under optimal conditions, this assay exhibited a working range of 0.04–19 ng/mL, with half-maximum inhibition (IC50) and limit of detection values of 0.27 and 0.02 ng/mL, respectively. After three sample pretreatment procedures were checked, a simple dilution method was adopted for further use. The stabilisation studies demonstrated that the icELISA kits can be stored for at least 180 to 240 days, at 4°C and −20°C, respectively. When applied to bovine samples, the data from icELISA and gas chromatography coupled to mass spectrometry showed excellent correlation (r2=0.9923 in muscle, 0.9884 in liver and 0.9957 in kidney). Therefore, this assay has the potential to be incorporated into a quantitative monitoring programme for the rapid screening of TES residue in foods.

Development of PVC and PP Resin Pellet Certified Reference Materials for Heavy Metal Analysis with Respect to the RoHS Directive

The PVC (polyvinyl chloride) and the PP (polypropylene) resin pellet certified reference materials (CRMs, NMIJ CRM 8123-a and 8133-a) with respect to the RoHS (restriction of the use of hazardous substances in electrical and electronics equipment) directive were developed in the present study. Both the sample pretreatment and the measurement procedures for the determination of Cd, Cr, Hg and Pb in the PVC and the PP resin pellets were examined, and several analytical methods were developed. Microwave acid digestion and dry-ashing followed by acid digestion procedures were examined as sample pretreatment procedures. In the case of measurement procedures, an inductively coupled plasma mass spectrometry (ICPMS) including an isotope dilution mass spectrometry (IDMS) and an ICP optical emission spectrometry (ICPOES) were used. Since the analytical results obtained by different analytical methods showed good agreement within their uncertainties estimated, it was concluded that the analytical methods established in this study were reliable with high accuracy. The homogeneity and the stability of Cd, Cr, Hg and Pb in these plastic CRMs were also evaluated, and the results revealed sufficient homogeneity of 0.3-0.7% and stability for more than 5 years. From these results, it is considered that these CRMs are useful CRM for the determination of Cd, Cr, Hg and Pb in plastics with respect to the RoHS directive.

Determination of 30 organochlorine pesticides in animal-originated food products using combined purification by gel permeation chromatography and solid-phase extraction coupled with gas chromatography-mass spectrometry

A new analytical method was developed for the determination of 30 organochlorine pesticides (OCPs) in animal-originated food, including pork, chicken, fish and shrimp. The combined purification by gel permeation chromatography (GPC) and solid-phase extraction (SPE) were established by optimizing different fraction collection times. The detection conditions can be achieved by gas chromatography-mass spectrometry (GC-MS) using selective ion monitoring (SIM). Isotopic internal standards were used for the quantitative determination of the 30 OCPs. The sample pretreatment procedure was based on acetonitrile extraction and combined purification of GPC and Florisil SPE cartridge. The experimental results showed that the linear ranges for 30 OCPs were 5.0 - 500.0 microg/L, the correlation coefficients were better than 0.996, and the method detection limits (MDLs) of the 30 OCPs were 0.2 - 2.7 microg/kg. The spiked recoveries at three levels of 5.0, 10.0 and 20.0 microg/kg using pork, chicken, fish and shrimp samples as blank matrices were in the range of 55.0% - 119.1%, the relative standard deviations (RSDs) were in the range of 0.4% - 15.0%. The method has the advantages of wide linear range, high sensitivity and efficient clean-up procedure, and consistent with the demand of pesticide routine analysis.

Monitoring of chronic Cannabis abuse: An LC–MS/MS method for hair analysis

An advanced analytical method based on liquid chromatography–tandem mass spectrometry (LC–MS/MS), has been developed for the identification and determination in hair of Δ9-tetrahydrocannabinol together with its major metabolite 11-nor-9-carboxy-Δ9-tetrahydrocannabinol. Since the latter is formed endogenously, it allows the assessment of chronic use excluding passive exposure to Cannabis. The sample pre-treatment procedure is based on a feasible incubative extraction followed by a liquid–liquid extraction step. Chromatographic separation was performed using a reversed-phase column and gradient elution with a formic acid/acetonitrile/water mobile phase. The limits of quantitation and of detection were 3pg/mg and 1pg/mg, respectively, for both analytes. The method was successfully applied to the analysis of hair samples from Cannabis abusers; the analyte concentrations found ranged from 55 to 100pg/mg for Δ9-tetrahydrocannabinol and from 5 to 10pg/mg for 11-nor-9-carboxy-Δ9-tetrahydrocannabinol. Accuracy studies also gave satisfactory results (recovery>87%), thus confirming the suitability of the assay for chronic consumption monitoring.

Considerations for U-series dating of sediments: Insights from the Flinders Ranges, South Australia

Uranium isotope ratios have been determined for the fine-grained detrital fraction of Pleistocene Wilkawillina valley-fill sediments, four local Proterozoic bedrock samples and fine-grained aeolian material from a sand dune deposit of the Flinders Ranges, South Australia. The aim was to quantify the comminution age, i.e. the time elapsed since physical weathering of the bedrock, and residence time of the valley-fill sediments and to place tighter constraints on input parameters for the comminution age calculation. Despite using two independent approaches for determination of the recoil lost fraction of 234U from the sediment (weighted geometric and surface area estimates), samples fail to produce realistic comminution ages and hence, residence times. The issues involved in the ability to determine sediment comminution ages are discussed. The (234U/238U) activity ratio of the local bedrock is not in secular equilibrium, despite the bedrock being much older than 1Ma, i.e. the timeframe for 234U and 238U to reach secular equilibrium in a closed system. Using the average Flinders Ranges bedrock (234U/238U) ratio instead of an assumed (234U/238U) activity ratio of unity for the source would significantly reduce calculated residence times. This result warrants concern for future studies using the comminution approach for which a secular equilibrium source (234U/238U) activity ratio is assumed. Significant input of aeolian material may modify the measured (234U/238U) activity ratios. Such input may be more tightly constrained in future studies using rare earth element and radiogenic isotopic data. Future comminution studies would benefit from further consideration of the importance of 1) leaching lost 234U from source rock and bulk sediment samples, 2) wind deposition of fine-grained material and 3) the appropriateness and robustness of sample pre-treatment procedures.

Dispersed particle extraction—A new procedure for trace element enrichment from natural aqueous samples with subsequent ICP-OES analysis

A novel sample pre-treatment method for multi trace element enrichment from environmental waters prior to optical emission spectrometry analysis with inductively coupled plasma (ICP-OES) is proposed, based on dispersed particle extraction (DPE). This method is based on the use of silica nanoparticles functionalized with strong cation exchange ligands. After separation from the investigated sample solution, the nanoparticles used for the extraction are directly introduced in the ICP for measurement of the adsorbed target analytes. A prerequisite for the successful application of the developed slurry approach is the use of sorbent particles with a mean size of 500nm instead of commercially available μm sized beads. The proposed method offers the known advantages of common bead-injection (BI) techniques, and further circumvents the elution step required in conventional solid phase extraction procedures. With the use of 14.4mL sample and addition of ammonium acetate buffer and particle slurry limits of detection (LODs) from 0.03μgL−1 for Be to 0.48μgL−1 for Fe, with relative standard deviations ranging from 1.7% for Fe and 5.5% for Cr and an average enrichment factor of 10.4 could be achieved. By implementing this method the possibility to access sorbent materials with irreversible bonding mechanisms for sample pre-treatment is established, thus improvements in the selectivity of sample pre-treatment procedures can be achieved. The presented procedure was tested for accuracy with NIST standard reference material 1643e (fresh water) and was applied to drinking water samples from the vicinity of Vienna.

OVERVIEW OF ANALYTICAL PROCEDURES FOR FATTY AND RESIN ACIDS IN THE PAPERMAKING PROCESS

This review describes the role of wood extractives, especially fatty and resin acids, in papermaking, as well as the importance of their removal from process waters. One of the main aims is also to illustrate versatile analysis methods for this purpose and highlight recent developments in corresponding applications. Most of the current methods require time-consuming and laborious sample pretreatment procedures prior to gas chromatography coupled either with flame ionization or mass selective detection. However, some faster, even online techniques with minimum sample pretreatment, are also available, mainly including high performance liquid chromatography coupled with mass spectrometry. The advantages and disadvantages of all analytical procedures are briefly discussed.

Rapid and Simple Analysis of Pesticides Persisting on Green Pepper Surfaces Swabbing with Solvent-Moistened Cotton

A rapid and simple nondestructive extraction (NDE) method that includes wiping off of systemic neonicotinoid insecticides has been developed to streamline sample pretreatment procedures conducted before chromatographic determination. Pesticide residues were extracted from green pepper surfaces by swabbing them with absorbent cotton moistened with acetone or acetonitrile. After spraying of pesticides, the extraction rate decreased gradually, except for thiacloprid. Presumably, extraction rates depend on the physicochemical properties of pesticides, especially water solubility. It was thought that the applicability of the proposed method greatly depended on the systemic speed of each pesticide, and water solubility was placed as the index that was important to making certain. Direct analysis of some insecticides persisting on sample surfaces has been possible only by extraction before chromatographic determination. These findings indicate strongly that the proposed NDE method has collateral conditions, but it appears promising for on-site pretreatment for pesticide residue analysis.

Electrokinetic injection across supported liquid membranes: New sample pretreatment technique for online coupling to capillary electrophoresis. Direct analysis of perchlorate in biological samples

A simple and sensitive method for quantifying perchlorate in biological samples using CE and capacitively coupled contactless conductivity detection was developed. An online combination of a supported liquid membrane, an inert polypropylene membrane impregnated with 1-hexanol, and electrokinetic injection of perchlorate across the supported liquid membrane directly into the separation capillary reduced the need for laborious sample pretreatment procedures, resulting in a cheap and rapid method with low LODs capability. Baseline separation of perchlorate and other anions in biological samples was achieved in background electrolyte solution consisting of 15 mM nicotinic acid and 1 mM 3-(N,N-dimethylmyristylammonio)propanesulfonate at pH 3.3. The analytical method showed excellent parameters in terms of reproducibility; RSD values for peak areas and corrected migration times at a spiked concentration of 100 μg/L of perchlorate were below 10 and 0.4%, respectively. Linear calibration curves were obtained for perchlorate in the concentration range 10-1000 μg/L (r(2) >0.999) with LODs between 2 and 5 μg/L for human urine, breast milk, serum, cow's milk, and red wine. Recoveries at 25 μg/L of perchlorate were between 97 and 106% for all biological samples. The low LODs rivaling those of presently used analytical methods support the use of this method for quantification of perchlorate in biological samples in the future.

Overcoming Bioanalytical Challenges in An Onglyza ® Intravenous [ 14 C]Microdose Absolute Bioavailability Study With Accelerator Ms

An absolute bioavailability study that utilized an intravenous [(14)C]microdose was conducted for saxagliptin (Onglyza(®)), a marketed drug product for the treatment of Type 2 diabetes mellitus. Concentrations of [(14)C]saxagliptin were determined by accelerator MS (AMS) after protein precipitation, chromatographic separation by UPLC and analyte fraction collection. A series of investigative experiments were conducted to maximize the release of the drug from high-affinity receptors and nonspecific adsorption, and to determine a suitable quantitation range. A technique-appropriate validation demonstrated the accuracy, precision, specificity, stability and recovery of the AMS methodology across the concentration range of 0.025 to 15.0 dpm/ml (disintegration per minute per milliliter), the equivalent of 1.91-1144 pg/ml. Based on the study sample analysis, the mean absolute bioavailability of saxagliptin was 50% in the eight subjects with a CV of 6.6%. Incurred sample reanalysis data fell well within acceptable limits. This study demonstrated that the optimized sample pretreatment and chromatographic separation procedures were critical for the successful implementation of an UPLC plus AMS method for [(14)C]saxagliptin. The use of multiple-point standards are useful, particularly during method development and validation, to evaluate and correct for concentration-dependent recovery, if observed, and to monitor and control process loss and operational variations.