Quantification of five neonicotinoids in human urine by modified QuEChERS with isotope dilution mass spectrometry: a preliminary study for the development of certified reference material

ABSTRACT

A study for the quantification method of neonicotinoid pesticides (Neos) in human urine that aims to develop the certified reference material (CRM) in the future was carried out. A modified Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) method based on dispersive solid phase extraction (d-SPE) with isotope dilution mass spectrometry (IDMS) was evaluated for the quantification of five Neos (acetamiprid, dinotefuran, imidacloprid, thiacloprid and thiamethoxam) in human urine control by optimisation of the extraction solvent, and the type and amount of d-SPE material. Two types of human urine controls (sample A and B) containing 5 μg/kg and/or 25 μg/kg of Neos were prepared and employed for this study. The results of spike test using sample A (2 mL) obtained by modified QuEChERS method with acetone extraction (10 mL) and d-SPE using a strong cation exchanger (silica gel modified with benzenesulphonic acid phase, SCX) 500 mg were as follows: 97.8%–103.1% for 5 μg/kg and 97.2%–102.7% for 25 μg/kg (described as percentage by the quantification results of IDMS relative to the spiked amount of pesticides). These results were comparable to those by SPE cartridge method using SCX functionalised hydrophilic styrene divinylbenzene (PCX). The repeatabilities of the analyses, represented as relative standard deviations, were also comparable for spike level 5 μg/kg and 25 μg/kg of Neos: 0.1%–7.2% for modified QuEChERS with IDMS and 0.2%–5.6% for PCX cartridge method. The results of spike test using sample B for a spike level 5 μg/kg were 96.0%–100.6%. These results indicate that modified QuEChERS method with IDMS can be used for the development of certified reference material and has the potential to use for the quantification of Neos in real human urine.     

Improving species-specific IDMS: the advantages of triple IDMS

Triple isotope dilution mass spectrometry (triple IDMS) has been applied for the first time on protein quantification, especially on transferrin. Transferrin as an acute phase protein is a marker for several inflammation processes in the human body. Therefore, in Germany, the accurate and precise measurement of this important analyte is required. In this work, a new approach to triple IDMS is described and compared to double IDMS. Also, complete uncertainty budgets for both methods were set up to demonstrate the ability of this method to be used as a reference procedure. The relative expanded uncertainty (k?=?2) for triple IDMS (3.6 %) is smaller than the one for double IDMS (4.0 %). The content of transferrin found in the human serum reference material ERM-DA470k/IFCC ((2.41?±?0.08) g/kg) with both methods was in good agreement with each other and with the certificate. For triple IDMS ((2.426?±?0.086) g/kg) and for double IDMS ((2.317?±?0.092) g/kg), transferrin was determined. Although triple IDMS is a little more time consuming compared to double IDMS, there is the advantage that the isotopic composition of the spike material does not have to be determined. This is very useful especially in case of a marginal isotopic enrichment in the spike or problems with the accurate measurement of the spike isotope ratio.

The analysis of thyroxine in human serum by an 'exact matching' isotope dilution method with liquid chromatography/tandem mass spectrometry

A method for the analysis of thyroxine in human serum, utilising 'exact matching' isotope dilution mass spectrometry (IDMS) in combination with liquid chromatography/tandem mass spectrometry (LC/MS/MS), has been developed with a limit of quantification of 0.5 ng g(-1) of thyroxine in human serum. The extraction and clean-up of thyroxine from serum involves an efficient protein-precipitation stage followed by a solid-phase extraction procedure to produce an extract essentially free from interfering compounds. The method is reproducible, with expanded uncertainties of less than 2%, and is relatively fast to perform.     

Determination of melamine in milk powder using gas chromatography-high-resolution isotope dilution mass spectrometry

A GC-high-resolution isotope dilution MS (IDMS) method for the quantification of melamine in milk powder is described. The developed technique is compared to the LC-IDMS/MS technique, typically used for the determination of melamine in various matrices. The accuracy of the GC-high-resolution IDMS method was demonstrated when a small degree of equivalence was obtained in a regional comparative study involving the determination of melamine in milk powder.     

Biosynthesis of 15N-labeled cylindrospermopsin and its application as internal standard in stable isotope dilution analysis

Cylindrospermopsin (CYN) is a cyanobacterial toxin associated with human and animal poisonings. Due to its toxicity in combination with its widespread occurrence, the development of reliable methods for selective, sensitive detection and accurate quantification is mandatory. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis using stable isotope dilution analysis (SIDA) represents an ideal tool for this purpose. U-[¹⁵N₅]-CYN was synthesized by culturing Aphanizomenon flos-aquae in Na¹⁵NO₃-containing cyanobacteria growth medium followed by a cleanup using graphitized carbon black columns and mass spectrometric characterization. Subsequently, a SIDA-LC-MS/MS method for the quantification of CYN in freshwater and Brassica matrices was developed showing satisfactory performance data. The recovery ranged between 98 and 103 %; the limit of quantification was 15 ng/L in freshwater and 50 μg/kg dry weight in Brassica samples. The novel SIDA was applied for CYN determination in real freshwater samples as well as in kale and in vegetable mustard exposed to toxin-containing irrigation water. Two of the freshwater samples taken from German lakes were found to be CYN-contaminated above limit of quantification (17.9 and 60.8 ng/L). CYN is systemically available to the examined vegetable species after exposure of the rootstock leading to CYN mass fractions in kale and vegetable mustard leaves of 15.0 μg/kg fresh weight and 23.9 μg/kg fresh weight, respectively. CYN measurements in both matrices are exemplary for the versatile applicability of the developed method in environmental analysis.      

Improved sample preparation of glyphosate and methylphosphonic acid by EPA method 6800A and time‐of‐flight mass spectrometry using novel solid‐phase extraction

The employment of chemical weapons by rogue states and/or terrorist organizations is an ongoing concern in the United States. The quantitative analysis of nerve agents must be rapid and reliable for use in the private and public sectors. Current methods describe a tedious and time‐consuming derivatization for gas chromatography–mass spectrometry and liquid chromatography in tandem with mass spectrometry. Two solid‐phase extraction (SPE) techniques for the analysis of glyphosate and methylphosphonic acid are described with the utilization of isotopically enriched analytes for quantitation via atmospheric pressure chemical ionization–quadrupole time‐of‐flight mass spectrometry (APCI‐Q‐TOF‐MS) that does not require derivatization. Solid‐phase extraction‐isotope dilution mass spectrometry (SPE‐IDMS) involves pre‐equilibration of a naturally occurring sample with an isotopically enriched standard. The second extraction method, i‐Spike, involves loading an isotopically enriched standard onto the SPE column before the naturally occurring sample. The sample and the spike are then co‐eluted from the column enabling precise and accurate quantitation via IDMS. The SPE methods in conjunction with IDMS eliminate concerns of incomplete elution, matrix and sorbent effects, and MS drift. For accurate quantitation with IDMS, the isotopic contribution of all atoms in the target molecule must be statistically taken into account. This paper describes two newly developed sample preparation techniques for the analysis of nerve agent surrogates in drinking water as well as statistical probability analysis for proper molecular IDMS. The methods described in this paper demonstrate accurate molecular IDMS using APCI‐Q‐TOF‐MS with limits of quantitation as low as 0.400 mg/kg for glyphosate and 0.031 mg/kg for methylphosphonic acid. Copyright © 2012 John Wiley & Sons, Ltd.     

Isotope pattern deconvolution-tandem mass spectrometry for the determination and confirmation of diclofenac in wastewaters

Isotope dilution mass spectrometry (IDMS) based on isotope pattern deconvolution (IPD) has been applied here to MS/MS (QqQ) in order to carry out the quantification and confirmation of organic compounds in complex matrix water samples without the use of a methodological IDMS calibration graph. In this alternative approach, the isotope composition of the spiked sample is measured after fragmentation by SRM and deconvoluted into its constituting components (molar fractions of natural abundance and labeled compound) by multiple linear regression (IPD). The procedure has been evaluated for the determination of the pharmaceutical diclofenac in effluent and influent urban wastewaters and fortified surface waters by UHPLC (ESI) MS/MS using diclofenac-d₄ as labeled compound. Calculations were performed acquiring a part and the whole fragment cluster ion, achieving in all cases recoveries within 90–110% and coefficients of variation below 5% for all water samples tested. In addition, potential false negatives arising from the presence of diclofenac-d₂ impurities in the labeled compound were avoided when the proposed approach was used instead of the most usual IDMS calibration procedure. The number of SRM transitions measured was minimized to three to make possible the application of this alternative technique in routine multi-residue analysis.     

Development of isotope dilution-liquid chromatography/mass spectrometry combined with standard addition techniques for the accurate determination of tocopherols in infant formula

During the development of isotope dilution-liquid chromatography/mass spectrometry (ID-LC/MS) for tocopherol analysis in infant formula, biased measurement results were observed when deuterium-labeled tocopherols were used as internal standards. It turned out that the biases came from intermolecular H/D exchange and intramolecular H/D scrambling of internal standards in sample preparation processes. Degrees of H/D exchange and scrambling showed considerable dependence on sample matrix. Standard addition-isotope dilution mass spectrometry (SA-IDMS) based on LC/MS was developed in this study to overcome the shortcomings of using deuterium-labeled internal standards while the inherent advantage of isotope dilution techniques is utilized for the accurate recovery correction in sample preparation processes. Details of experimental scheme, calculation equation, and uncertainty evaluation scheme are described in this article. The proposed SA-IDMS method was applied to several infant formula samples to test its validity. The method was proven to have a higher-order metrological quality with providing very accurate and precise measurement results.     

Determination of Chloramphenicol in Milk Using a QuEChERS-Based on Liquid Chromatography Tandem Mass Spectrometry Method

A quantitative method for the determination of chloramphenicol in milk samples was developed based on the QuEChERS (quick, easy, cheap, effective, rugged, and safe) approach for liquid chromatography–tandem mass spectrometry (LC–MS/MS). Homogenized milk samples were extracted with acetonitrile. The partitioning step was performed after the addition of magnesium sulfate and sodium chloride. Chloramphenicol was determined using the electrospray negative ionization mode with tandem mass spectrometry. The procedure was validated according to the requirements of Commission Decision 2002/657/EC. The apparent recovery ranged from 90% to 110% and within-laboratory reproducibility was lower than 12%. The calculated limit of decision was 0.10 μg kg^?1 and the detection capability was 0.15 μg kg^?1. Validation results demonstrated that this method fulfills criteria for the determination of chloramphenicol in milk.     

Comparison of orthogonal liquid and gas chromatography–mass spectrometry platforms for the determination of amino acid concentrations in human plasma

Concentrations of amino acids in a human plasma pool were determined using four independent quantification methods. Orthogonal separation schemes (LC, GC, or GC×GC) and detection systems (triple quadrupole or time-of-flight mass spectrometry) are shown to demonstrate excellent consistency among platforms for quantifying 18 amino acids in NIST Standard Reference Material (SRM) 1950 Metabolites in Human Plasma using a well-characterized isotope dilution (ID) quantification method. Measured levels were consistent with reference values in plasma from the literature. Individual amino acid concentrations in plasma varied by over an order of magnitude ranging from 1.83 μg/g to 28.0 μg/g (7.78 μmol/L to 321 μmol/L). Average variability (coefficient of variation) between experimental amino acid concentrations (excluding cysteine) among all methods was 6.3%. Certified mass fraction values for amino acids in NIST SRM 1950 will be established from statistically weighted means of all experimental results.     

Achieving traceable chemical measurements: inter-laboratory evaluation of a simplified technique for isotope dilution mass spectrometry. Part 2. Methodology for high accuracy analysis of organic analytes

A high accuracy measurement procedure developed and validated at LGC has been transferred to a number of expert UK laboratories, and their experience in applying the technique has been evaluated by inter-laboratory comparisons. It is an “exact matching” calibration procedure for analysis of organic analytes using isotope dilution mass spectrometry (IDMS). This calibration procedure uses a calibration blend and a sample blend with closely matched isotope amount ratios, and is an iterative process, culminating in the calibration blend and sample blend having identical isotope amount ratios. It is capable of high accuracy, since systematic errors in the determination of the isotope amount ratios are cancelled out. A series of four inter-laboratory comparisons of increasing difficulty were carried out involving a number of expert laboratories. The first three comparisons used gas chromatography mass spectrometry (GC–MS) analysis of the pesticide metabolite (pp′-dichlorodiphenyl) dichloroethylene (pp′-DDE), involving both conventional calibration and IDMS exact matching procedures for pp′-DDE in a solvent and a complex liquid matrix (corn oil). The fourth comparisons utilised liquid chromatography mass spectrometry (LC–MS) and involved the analysis of sulphamethazine (4-amino-N-(4,6 dimethyl-2 pyrimidinyl) benzenesulphonamide) in solvent using IDMS and conventional calibration techniques. Following the first trial, a workshop for participants was held on the use of the exact matching procedure together with a short course on uncertainty estimation. The results of the comparisons clearly showed the superior accuracy of using IDMS with the exact matching procedure for both GC–MS and LC–MS applications. These comparisons and the workshop have enabled the methodology to be transferred to UK industry, helping to improve UK measurement capability.

     

Determination of Urea in Milk by Liquid Chromatography-Isotope Dilution Mass Spectrometry

A definitive method based on liquid chromatography isotope dilution mass spectrometry (LC-IDMS) has been developed for the determination of milk urea, an indicator of nutrition status for the lactating animals. The milk samples were treated twice by sequentially adding acetonitrile and chloroform to precipitate proteins and then were directly separated using normal phase liquid chromatography without chemical derivatization. After the matrix separation, exact matching IDMS was used for the determination of milk urea, with high accuracy, high precision, good linearity and low uncertainty. The recoveries obtained for the four spiked milk samples were 100.6–102.2%. The linear range of signal responses was 10–2000 mg · kg^?1 with a linearity coefficient of 0.9995. The intraday and interday precisions in terms with relative standard deviations (RSDs) were 0.17–0.38% and 0.28–0.40%, respectively. The uncertainties of the whole sample analysis process were estimated to be 0.83%, 0.60%, and 0.64% for three samples with concentrations of 151.28, 184.36, and 266.66 mg · kg^?1.     

Achieving traceable chemical measurements: inter-laboratory evaluation of a simplified technique for isotope dilution mass spectrometry. Part 2. Methodology for high accuracy analysis of organic analytes

A high accuracy measurement procedure developed and validated at LGC has been transferred to a number of expert UK laboratories, and their experience in applying the technique has been evaluated by inter-laboratory comparisons. It is an “exact matching” calibration procedure for analysis of organic analytes using isotope dilution mass spectrometry (IDMS). This calibration procedure uses a calibration blend and a sample blend with closely matched isotope amount ratios, and is an iterative process, culminating in the calibration blend and sample blend having identical isotope amount ratios. It is capable of high accuracy, since systematic errors in the determination of the isotope amount ratios are cancelled out. A series of four inter-laboratory comparisons of increasing difficulty were carried out involving a number of expert laboratories. The first three comparisons used gas chromatography mass spectrometry (GC–MS) analysis of the pesticide metabolite (pp′-dichlorodiphenyl) dichloroethylene (pp′-DDE), involving both conventional calibration and IDMS exact matching procedures for pp′-DDE in a solvent and a complex liquid matrix (corn oil). The fourth comparisons utilised liquid chromatography mass spectrometry (LC–MS) and involved the analysis of sulphamethazine (4-amino-N-(4,6 dimethyl-2 pyrimidinyl) benzenesulphonamide) in solvent using IDMS and conventional calibration techniques. Following the first trial, a workshop for participants was held on the use of the exact matching procedure together with a short course on uncertainty estimation. The results of the comparisons clearly showed the superior accuracy of using IDMS with the exact matching procedure for both GC–MS and LC–MS applications. These comparisons and the workshop have enabled the methodology to be transferred to UK industry, helping to improve UK measurement capability.     

Development of melamine certified reference material in milk using two different isotope dilution mass spectrometry techniques

A new certified reference material (CRM) of melamine in milk GLHK-11-02 was developed aiming to address the great demand from the testing community after the melamine crises. The material was prepared by adding an appropriate quantity of melamine into the skimmed milk samples and the final product was in the form of fine lyophilized powder. Characterization of the material relied on two newly developed gravimetric isotope dilution mass spectrometry (IDMS) methods, one using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and another gas chromatography-mass spectrometry (GC-MS). Experimental parameters with crucial effects on the performance of the two IDMS methods were thoroughly investigated. These included purity of standard used, equilibration time of isotopes, efficiency of extraction methods as well as possible interferences from the matrix and melamine analogues. Precision was found to be excellent with a coefficient of variation of 2.5% for the LC-IDMS/MS (n=46) and 1.9% for the GC-IDMS (n=30) respectively. Using one-tail Student's t-test at 95% confidence interval, analytical data sets generated from the two methods were found to exhibit no significant difference. Measurement accuracy of the methods was further verified through an Asia Pacific Metrology Program (APMP) pilot study. Analytical results of the present LC-IDMS/MS for the two milk test samples at the concentration level of about 0.45 and 3.5 mg kg(-1) were proven to be very good. There were excellent overlaps between our results and the assigned reference values, and the absolute deviation was less than 3.2%. Both the LC-IDMS/MS and GC-IDMS methods were shown to be sufficiently reliable and accurate for certification of the melamine CRM. Certified value of melamine in dry mass fraction in GLHK-11-02 was 1.14 mg kg(-1). Expanded uncertainty due to sample inhomogeneity, long term and short term stability and variability in the characterization procedure was at 7.1% or 0.08 mg kg(-1). The CRM is primarily used to provide a complete method validation for and to improve the technical competence of melamine analysis to food and chemical testing laboratories.     

High yield synthesis and characterization of isotopically enriched monoethylmercury chloride (C2H5201HgCl)

An important but commercially unavailable compound isotopically enriched monoethylmercury chloride (C₂H₅²⁰¹HgCl), has been synthesized from commercially available ²⁰¹HgO (98.11% enriched isotopic purity) and tetraethyltin. The required synthesis time is 1 h at 90 °C, and the product is the single product of monoethylmercury chloride, yielding more than 95% as ²⁰¹Hg in C₂H₅²⁰¹Hg⁺ (98.19 ± 0.22% enriched isotopic purity). The synthesized product was analyzed with high‐performance liquid chromatography coupled with inductively coupled plasma mass spectrometry (HPLC‐ICP‐MS) to determine its concentration, isotopic composition and purity. The synthetic isotopically enriched monoethylmercury synthesized can be used in speciated isotope dilution mass spectrometry (SIDMS) and isotope dilution mass spectrometry (IDMS) analyses as a standard. Copyright © 2005 John Wiley & Sons, Ltd.     

Advancement of isotope separation for the production of reference standards

Idaho National Laboratory (INL) operates a mass separator that is currently producing high purity isotopes for use as internal standards for high precision isotope dilution mass spectrometry (IDMS). In 2008, INL began the revival of the vintage 1970s era instrument. Advancements thus far include the successful upgrading and development of system components such as the vacuum system, power supplies, ion-producing components, and beam detection equipment. Progress has been made in the separation and collection of isotopic species including those of Ar, Kr, Xe, Sr, and Ba. Particular focuses on ion source improvements and developments have proven successful with demonstrated output beam currents of over 10 μA ¹³⁸Ba and 350 nA ¹³⁴Ba from a natural abundance Ba source charge (~2.4 % ¹³⁴Ba). In order to increase production and collection of relatively high quantities (mg levels) of pure isotopes, several improvements have been made in ion source designs, source material introduction, and ion detection and collection. These improvements have produced isotopes of high purity (>98 %) and in quantities in the tens of micrograms per run. The instrument and results for pure isotope production for IDMS standards will be presented.     

Towards compound-independent calibration for organic compounds using online isotope dilution mass spectrometry

Isotope dilution mass spectrometry (IDMS) can be considered a primary measurement method directly traceable to the International System of Units (SI). This measurement technique is increasingly employed in routine laboratories, owing to its unequalled analytical performance, precision and ease of accreditation. Unfortunately, for the adequate application of IDMS, several isotopically labelled standards, corresponding to the compounds of interest, are required. Additionally, when the enriched isotope is continuously added after a chromatographic separation, and an elemental ion source is used, it allows quantification of the different analytes being eluted from the column without requiring specific standards for each compound (online IDMS). In this article, we discuss how the traditional applicability of online IDMS for elemental speciation can be dramatically expanded by using carbon isotope tracers, oxidation or combustion reactions and a conventional molecular ion source. With such a strategy every carbon-containing compound being eluted from a chromatography system can be quantified without the need for specific standards as long as quantitative combustion/oxidation and complete elution occur. So far, only gas chromatography–combustion–mass spectrometry applications have been described, but recent results indicate the great possibilities of extending this novel approach to the quantification of organic compounds after separation by liquid chromatography.     

Development of a candidate certified reference material of cypermethrin in green tea

This paper presents the preparation of a candidate certified reference material (CRM) of cypermethrin in green tea, GLHK-11-01a according to the requirements of ISO Guide 34 and 35. Certification of the material was performed using a newly developed isotope dilution mass spectrometry (IDMS) approach, with gas chromatography high resolution mass spectrometry (GC–HRMS) and gas chromatography–tandem mass spectrometry (GC–MS/MS). Statistical analysis (one-way ANOVA) showed excellent agreement of the analytical data sets generated from the two mass spectrometric detections. The characterization methods have also been satisfactorily applied in an Asia-Pacific Metrology Program (APMP) interlaboratory comparison study. Both the GC–HRIDMS and GC–IDMS/MS methods proved to be sufficiently reliable and accurate for certification purpose. The certified value of cypermethrin in dry mass fraction was 148 μg kg⁻¹ and the associated expanded uncertainty was 14 μg kg⁻¹. The uncertainty budget was evaluated from sample in homogeneity, long-term and short-term stability and variability in the characterization procedure. GLHK-11-01a is primarily developed to support the local and wider testing community on need basis in quality assurance work and in seeking accreditation.     

Multiclass Compatible Sample Preparation for UHPLC–MS/MS Determination of Aflatoxin M1 in Raw Milk

A sample preparation method for aflatoxin M1 (AFM1) determination in raw milk was optimized following the quick, easy, cheap, effective, rugged and safe (QuEChERS) strategy, as an alternative to the classic immunoaffinity column clean-up (IAC). The method was adapted to address the complexity of the milk matrix, and to be suitable for final determination by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC–MS/MS). This approach proved also to be compatible with the simultaneous extraction of pesticide residues and other contaminants (mycotoxins). Regarding AFM1, satisfactory linearity was achieved and appropriate sensitivity was maintained, using matrix-matched calibration to compensate for the heavy ion suppression. The accuracy and precision, which were determined through recovery studies, were 70–95 %, with the relative standard deviation below 15 % in all of the cases. The limit of detection (LOD, 0.002 μg L⁻¹) and limit of quantification (0.007 μg L⁻¹) are compatible with current worldwide regulations (maximum levels of 0.5 and 0.05 μg L⁻¹). The procedure was applied to samples that were naturally contaminated with a range of AFM1 at LOQ–0.187 μg L⁻¹, with comparable results to IAC clean-up, which was employed as a reference method. Therefore, AFM1 determination in raw milk by UHPLC–MS/MS detection through the present QuEChERS extraction constitutes a reliable alternative to IAC clean-up and exhibits advantages related to cost, accessibility of materials and simplicity of operation.

     

Combination of pentafluorophenylhydrazine derivatization and isotope dilution LC-MS/MS techniques for the quantification of apurinic/apyrimidinic sites in cellular DNA

Apurinic/apyrimidinic (AP) sites are common DNA lesions arising from spontaneous hydrolysis of the N-glycosidic bond and base-excision repair mechanisms of the modified bases. Due to the strong association of AP site formation with physically/chemically induced DNA damage, quantifying AP sites provides important information for risk assessment of exposure to genotoxins and oxidative stress. However, rigorous quantification of AP sites in DNA has been hampered by technical problems relating to the sensitivity and selectivity of existing analytical methods. We have developed a new isotope dilution liquid chromatography–coupled tandem mass spectrometry (LC-MS/MS) method for the rigorous quantification of AP sites in genomic DNA. The method entails enzymatic digestion of AP site-containing DNA by endo- and exonucleases, derivatization with pentafluorophenylhydrazine (PFPH), addition of an isotopically labeled PFPH derivative as internal standard, and quantification by LC-MS/MS. The combination of PFPH derivatization with LC-MS/MS analysis on a triple quadrupole mass spectrometer allows for sensitive and selective quantification of AP sites in DNA at a detection limit of 6.5 fmol, corresponding to 4 AP sites/10⁹ nt in 5 μg of DNA, which is at least ten times more sensitive than existing analytical methods. The protocol was validated by AP site-containing oligonucleotides and applied in quantifying methyl methanesulfonate-induced formation of AP sites in cellular DNA.