Isotope-dilution liquid chromatography-tandem mass spectrometry for sensitive quantification of human insulin in serum using derivatization-technique

Analytical Biochemistry 537 (2017) 26e32

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Isotope-dilution liquid chromatography-tandem mass spectrometry for sensitive quantification of human insulin in serum using derivatization-technique Yohei Sakaguchi*, Tomoya Kinumi, Akiko Takatsu Bio-medical Standards Group, Research Institute for Material and Chemical Measurement, National Metrology Institute of Japan (NMIJ), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba C-3, Ibaraki 305-8563, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 June 2017 Received in revised form 11 August 2017 Accepted 27 August 2017 Available online 30 August 2017

An isotope-dilution mass spectrometry (IDMS) method for measuring insulin levels in human serum was developed using C-terminal-derivatization method coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The carboxyl groups of Glu-C-cleavage products were derivatized with 1-(2pyrimidinyl)piperazine to increase MS/MS sensitivity and IDMS quantification, resulting in increases in LC-MS/MS peak areas of derivatized Glu-C-cleavage products of human insulin by ~23-(A5e17 peptide) to 49-fold(B14e21 peptide), respectively, as compared with results observed in the absence of derivatization. Separation was achieved on a C18 column by gradient elution at 0.3 mL/min, with a mobile phase composed of 0.1% formic acid in acetonitrile and water. Validation studies of target peptides (B1 e13 peptide and B14e21 peptide) revealed a linear response in the range of 0.05 ng/mL to 10 ng/mL (regression coefficient, r2 ¼ 0.9987 and 0.9988, respectively), a relative standard deviation within and between days of <8.6%, and spike and recovery test results indicating mean recoveries ranging from 100.2% to 106.6%. Comparison with an established commercial immunoassay showed high correlation (r2 ¼ 0.9943 and 0.9944, B1e13 peptide and B14e21 peptide, respectively) at serum concentrations of between 0.20 ng/mL and 1.51 ng/mL. These findings suggested that this IDMS-based approach was able to quantify human serum insulin with high sensitivity and precision in the reference interval and indicated a potential for determining serum-insulin reference-measurement procedures to allow traceable measurement. © 2017 Elsevier Inc. All rights reserved.

Keywords: Human insulin LC-MS/MS Derivatization Sensitive IDMS

Introduction Human insulin is a hormone produced by beta cells of pancreatic islets and plays important roles in the metabolism of carbohydrates by promoting the absorption of glucose from the blood into fat, liver, and skeletal muscle cells. The structure of human insulin consists of a two-peptide chain (A chain: 21 amino acids; B chain: 30 amino acids) connected by two disulfide bonds, with proinsulin cleavage by proteases producing a C-peptide and insulin [1,2]. The measurement of human insulin in serum allows the assessment of levels of endogenous insulin produced by the pancreas and insulin resistance. As routine laboratory tests, immunoassays, such as radioimmunoassays and enzyme-linked

* Corresponding author. E-mail address: [email protected] (Y. Sakaguchi). http://dx.doi.org/10.1016/j.ab.2017.08.019 0003-2697/© 2017 Elsevier Inc. All rights reserved.

immunosorbent assays, have been used to quantify levels of human insulin. These methods are capable of easily and sensitively quantifying human insulin in serum; however, the lack of reference-measurement procedures affects comparison of results between immunoassays during routine human-insulin measurement. Recently, isotope-dilution mass spectrometry (IDMS) was developed to establish a reliable analytical method enabling acquisition of traceable measurements for bioanalysis. Because serum consists of a complex mixture of numerous proteins, peptides, lipids, salts, and other ingredients, it is important to establish a proper method capable of isolating the analyte from biological fluid to enable successful IDMS measurement. In a previous study, we developed a method for quantifying human serum C-peptide levels via IDMS that successfully enabled traceable measurement [3]. The typical concentration of insulin in healthy human serum is below the ng/mL level, and in the case of diabetes patients, this

Y. Sakaguchi et al. / Analytical Biochemistry 537 (2017) 26e32

Abbreviations BSA DMF TT EDC ESI FIA HOAt IDMS LC-MS LOD MS/MS PP SRM

bovine serum albumin N,N-dimethylformamide dithiothreitol 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydrochloride electrospray ionization flow-injection analysis 1-hydroxy-7-azabenzotriazole isotope-dilution mass spectrometry liquid chromatography-mass spectrometry limit of detection tandem mass spectrometry 1-(2-pyrimidinyl)piperazine selected-reaction monitoring

level tends to be even lower due to the minimal secretion of insulin from beta cells of pancreatic islets [4]. To identify types of diabetes, measurement of insulin at low concentrations is required; therefore, a sensitive method for insulin quantification is needed. Previous studies reported successful application of liquid chromatography-mass spectrometry (LC-MS) for measuring insulin, as well as insulin preparations, in serum [5e9]. In these studies, in order to quantify low concentration of the human insulin in serum on LC-MS analysis, solid-phase extraction (SPE) [8,9] or immunoaffinity purification [5,6] was used for enrichment at sample preparation. However, derivatization method for sensitive analysis has never been used for insulin quantification using LC-MS analysis. Derivatization method can be combined with other enrichment sample preparations such as SPE and immunoaffinity purification and it is useful tool for quantification of low concentration insulin. In this study, we combined IDMS with derivatization methods to enable a more accurate quantification of insulin in order to achieve high sensitivity LC-MS analysis. Carboxyl-group derivatization methods were described previously [10] and involve addition of 1(2-pyrimidinyl)piperazine (PP) moieties to insulin to promote protonation for electrospray ionization (ESI). Here, we added insulin-D40 to the serum sample and standard solution to derive calibration curves prior to enzymatic digestion of Glu-C, derivatization, and LC-tandem MS (LC-MS/MS) analysis. By adding this, it is possible to cancel errors caused by experimental procedures after the addition or MS/MS detection. After optimization of the enzymatic reaction and derivatization, we assessed recovery of pretreatment during serum analysis, linearity of the calibration curve, and the reproducibility of results and compared the results of our method with those obtained from immunoassays to determine validity. As a calibrant of human insulin on serum analysis, the human insulin solution which was quantified by amino acid analysis using amino acid certified reference material was used to find the accurate concentration of calibrant of human insulin. The
me Inconcentration of this calibrant has traceability to SI (Syste ) and it is enabled traceable measurement of ternational d’Unite human insulin in serum.

27

(DMF), 4-methylmorpholine, 1-hydroxy-7-azabenzotriazole (HOAt), V8 protease (Glu-C), dithiothreitol (DTT), 2iodoacetamide, and PP were purchased from Wako Pure Chemical (Osaka, Japan). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydrochloride (EDC) was purchased from Dojindo Laboratories (Kumamoto, Japan). 2-Picoline borane was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Formic acid was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Formic acid and acetonitrile were LC-MS grade. Ultrapure water, purified using a Milli-Q system (Millipore, Billerica, MA, USA), was used to prepare all aqueous solutions. Human insulin-free serum and spiked human-insulin stock solution were purchased from Scipac, Ltd. (Sittingbourne, UK). Bovine serum albumin (BSA) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in water to 0.1% (v/v). Human insulin and human insulin-D40 were purchased from Peptide institute, Inc. (Osaka, Japan) and dissolved in 20 mM ammonium bicarbonate for stock solution. As a calibrant solution of human insulin, the stock solution of human insulin was diluted with 0.1% (v/v) BSA. The hydrogen atoms in the leucine of the B chain of human insulin-D40 (B6, B11, B15, and B17) was replaced with deuterium (Fig. 1). The mixtures used for the optimization study and protein quantification were gravimetrically prepared. Five batches of human pooled serum were provided by Tsukuba Medical Center Hospital (Ibaraki, Japan) and stored at 80  C. Insulin concentrations in each pooled serum sample were measured by chemiluminescence enzyme immunoassay using Lumipulse (Fujirebio, Tokyo, Japan). Instrumentation and conditions LC analysis was performed on a Nexera HPLC system (Shimadzu) equipped with a system controller (CBM-20A), a binary solventdelivery system (LC-30AD), a single-solvent-delivery system (LC20AD), an auto sampler (SIL-30AC), and a column heater (CTO20A). LabSolutions LC-MS software (Shimadzu) was used to control the instruments and to process the data. An Xselect CSH C18 column (50  2.1 mm i.d.; Waters Corp., Milford, MA, USA) was used for separation. The mass spectrometer used in this study was an LCMS-8040 triple quadrupole (Shimadzu) equipped with an ESI interface. The ESI mass spectrometer was operated in positive-ionization mode and in either scan, single-ion monitoring (precursor-ion scan), or selected-reaction monitoring (SRM) modes. Sample-solution preparation for quantification Standard human insulin and human insulin-D40 stock solutions were stored in polypropylene vials at 80  C until the day of analysis. The human insulin stock solution was quantified by amino acid analysis using amino acid certified-reference material (Laspartic acid, NMIJ CRM 6027-a; L-glutamic acid, NMIJ CRM 6026-a; L-valine, NMIJ CRM 6015-a; L-isoleucine, NMIJ CRM 6013-a; Lleucine, NMIJ CRM 6012-a; L-phenylalanine, NMIJ CRM 6014-a; National Meteorology Institute of Japan, Ibaraki, Japan). The human insulin stock solution was hydrolyzed by hydrochloric acid, and the resulting hydrolysate was employed in amino acid analyses by IDMS using isotopically labeled amino acids. Hydrolysis and amino acid analyses were conducted by gas-phase hydrolysis, and the hydrolyzed amino acids were quantified as previously described [11].

Materials and methods Preparation of serum and standard samples Reagents and materials Acetonitrile, 2-propanol, acetaldehyde, N,N-dimethylformamide

Sample blends were prepared gravimetrically by mixing a serum sample with the human insulin-D40 solution. Calibration

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Y. Sakaguchi et al. / Analytical Biochemistry 537 (2017) 26e32

Fig. 1. Schematic describing the method presented in this study.

blends were prepared gravimetrically by mixing standard human insulin and human insulin-D40 solutions. Acetnitrile (400 mL) was added to 100 mL of serum-sample blend or -calibration blend in a 5.0-mL polypropylene tube, and after vortex mixing for several seconds, the mixture was immediately centrifuged at 16,000g for 10 min at 4  C, and the supernatant was passed through a disposable filter (0.20 mm, i.d. 13 mm, polytetrafluoroethylene; Advantec Toyo, Tokyo, Japan). Thereafter, a portion of the filtrate was evaporated under reduced pressure and subjected to enzymatic digestion. Enzymatic digestion Endoprotease Glu-C was dissolved in 20 mM ammonium bicarbonate solution at 1 mg/mL, and the 100 mL aliquots were prepared and stored at 80  C as the stock solution. To residue obtained by preparation of serum sample or standard sample, 100 mL of 20 mM ammonium bicarbonate solution and 10 mL of 1 mg/mL Glu-C solution were added, and after vortex mixing for several seconds, the mixture was incubated for 24 h at 37  C. The resulting mixture was reduced by the addition of 20 mL of DTT solution (100 mM DTT in 20 mM ammonium bicarbonate solution) and incubation for 30 min at 50  C, followed by alkylation by adding 20 mL of iodoacetamide solution (240 mM iodoacetamide in 20 mM ammonium bicarbonate solution) and incubation for 15 min at room temperature in the dark. Excess reagent was inactivated by adding 20 mL of DTT solution (500 mM DTT in 20 mM ammonium bicarbonate solution). Derivatization procedure for Glu-C digested peptides The digested sample or standard mixture was evaporated under reduced pressure, followed by addition of 500 mL of 50% (v/v) 2propanol, 200 mL acetaldehyde, 10 mL 4-methylmorpholine, and 100 mL 2-picoline borane (200 mM in 2-propanol). After mixing, the obtained solutions were incubated at room temperature for 30 min, followed by drying under reduced pressure. We added 40 mL PP (400 mM in DMF), 40 mL HOAt [400 mM in DMF and 5% (v/v) 4methylmorpholine], and 40 mL EDC (400 mM in DMF) to the obtained residues, and after mixing, the samples were heated at 60  C

for 30 min. After the reaction, the obtained solutions were dried under reduced pressure and dissolved in 150 mL water. Sensitivity comparison To examine sensitivity of this derivatization method on LC-MS analysis, comparisons of measurements of intact peptides with those of amino-group- and carboxyl-group-derivatized peptides were performed using flow-injection analysis [FIA; mobile phase, 0.1% (v/v) formic acid in 50% (v/v) acetonitrile; flow rate, 0.05 mL/ min; and injection volume, 5 mL] and MS (selected-ion monitoring). A 1 mM standard solution containing intact peptides and a 1 mM solution containing derivatized peptides were diluted 100-fold with 0.1% (v/v) formic acid in 50% (v/v) acetonitrile, respectively, followed by mixing to a 1:1 ratio and analysis by FIA-MS. The derivatization procedure and MS conditions were the same as described earlier in this section. LC-MS/MS measurement and quantification of human insulin in serum B1e13 and B14e21 digested peptides were used for quantification, because these digested peptides contain leucine residues wherein the hydrogen has been replaced with deuterium (Fig. 1) in the insulin-D40. The LC conditions were as follows. Solvents A [0.1% (v/v) formic acid in water] and B [0.1% (v/v) formic acid in acetonitrile] were used as the mobile phases for gradient elution. The column oven temperature was set at 50  C, the injection volume was set at 5 mL, and total flow rate of pumps A and B was 0.3 mL/ min. The concentration of the mobile phase B was assigned to change linearly from 1% to 40% from 0 min to 10 min and in 1% increments from 10 min to 20 min. The MS conditions were set as follows: ion spray voltage, 5.0 kV; desolvation line temperature, 150  C; heat-block temperature, 500  C; drying gas, 5.0 L/min; nebulizer gas, 3.0 L/min; and collision-induced dissociation (CID) gas, 350 kPa. The precursor ions, product ions, and CID energies obtained with the SRM method for the derivatized peptides are shown in Table 1. Peak areas were integrated automatically and used to quantify peptide concentration. The concentration of each peptide was calculated using the calibration curves and the peak-

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Table 1 Precursor and product ions (Q1 and Q3) and collision energies for the SRM method using derivatized peptides. Digested peptide

Sequence

Precursor ion (m/z)

Product ion (m/z)

Collision energy (CE, eV)

B1-13 peptide Isotopically labeled B1-13 peptide (Human insulin-D40) B14-21 peptide Isotopically labeled B14-21 peptide (Human insulin-D40)

F(bis-ethly)VNQHLCGSHLVE (2PP) F(bis-ethly)VNQHL(d10)CGSHL(d10)VE (2PP) A(bis-ethly)LYLVCGE(2PP) A(bis-ethly)L(d10)YL(d10)VCGE(2PP)

630.0 636.7 636.9 646.9

176.2 176.2 172.1 172.1

47 47 48 48

area ratio of natural human insulin to human insulin-D40. To quantify the peptides in the human serum samples, calibrationstandard solutions at between 0.05 ng/mL and 10 ng/mL (0.05, 0.1, 0.5, 1, 5, and 10 ng/mL) were prepared by diluting the stock solutions. Validation study To confirm the feasibility of this analytical method for quantitative analysis, a validation study was performed using standard solution. All samples used for the validation study were subjected to derivatization after preparation by the procedure described as follows. The intraday precision values were assessed by performing each analysis six times on the same day. The limits of detection (LODs) were defined as the concentrations providing signal-tonoise ratios of three. In this study, in order to confirm that accurate quantification could be performed by using IDMS, recovery was determined based on the spiked value obtained from microbalance and the quantification value obtained from used of the method presented here. Spiked human insulin-free serum samples at two concentrations (10 ng/mL and 1 ng/mL serum) were prepared and analyzed using the present analytical method. Results and discussion Optimization of enzymatic digestion and derivatization-reaction conditions Enzyme-digestion conditions involving Glu-C were optimized using insulin-free serum spiked with insulin (0.1 mg/mL) and Glu-C concentrations ranging from 0.1 mg/mL to 10 mg/mL. Our results indicated that a Glu-C concentration of 1 mg/mL was optimal, and analyses of reaction times between 30 min and 48 h showed that

24 h was optimal. Acetaldehyde and PP were used as the derivatization reagents based on their successful use in analyzing low molecular weight peptides [10]. We used the same derivatization conditions as those used for low molecular weight peptide analysis, because the digested peptide obtained from human insulin were almost same molecular weight as peptides described in a previous study [10]. We determined that the optimal derivatization conditions for amines involved addition of 200 mL acetaldehyde, 10 mL 4methylmorpholine, and 100 mL 2-PB (200 mM in 2-propanol) to the sample and reaction for 30 min at room temperature. The optimal derivatization conditions for carboxyl groups involved addition of 40 mL PP (400 mM in DMF), 40 mL HOAt [400 mM in DMF and 5% (v/v) 4-methylmorpholine], and 40 mL EDC (400 mM in DMF) to the sample and reaction for 30 min at 60  C.

MS-detection conditions All derivatives were ionized via ESI on the positive-ionization mode, as protonated ions ([M þ 2H]2þ and [M þ 3H]3þ) from the expected structures. We selected the most intense fragment ions obtained from the protonated ions to represent precursor ions under the examined CID conditions. Although other fragment ions associated with the derivatized peptides were observed in the MS/ MS spectra, the most intense precursor-product transitions were used as the quantification transitions in SRM mode to obtain the most sensitive quantitative analysis of the peptides. Other MS/MSdetection conditions were also optimized to obtain the highest peak intensities for the derivatized peptides. The obtained optimized SRM conditions are shown in Table 1 and typical SRM chromatograms are shown in Fig. 2.

Fig. 2. Typical SRM chromatograms for the 10 ng/mL standard solutions of human insulin using the present method.

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Table 2 Calibration curve linearity, repeatability, and LODs associated with the derivatized peptides analyzed using the method presented in this study. Digested peptide

Coefficients of determination (r2)a

Repeatabilityb (%, n ¼ 6)

Detection limitc (pmol/L)

B1e13 peptide B14e21 peptide

0.9987 0.9988

3.9 4.3

17 6.2

a b c

Calibration curves in the range of 0.05e10 ng/mL. Relative standard deviation of the peak area for 1 ng/mL. Defined as the amount per 5-mL injection volume yielding a signal-to-noise ratio of three.

Comparison analysis to determine sensitivity To demonstrate the sensitivity of this method, we compared the measurements of intact-digested peptides from human insulin with those of amino-group- and carboxyl-group-derivatized digested peptides from human insulin. The derivatized-peptide and intact-peptide solutions were mixed at a 1:1 ratio and analyzed by FIA-MS. The ratios of absolute ion abundances were compared for the derivatized peptides and intact peptides to determine the change in ESI response due to the derivatization. The ratios of the signals associated with the derivatized peptides relative to signals of the intact peptides were as follows: A1e4 peptide, 24.7; A5e17 peptide, 23.2; A18e2 peptide, 43.3; B1e13 peptide, 41.1; B14e21 peptide, 49.0; and B22e30 peptide, 33.4. For all examined peptides, we observed a >23-fold enhancement in ESI response from derivatized peptides as compared with nonderivatized intact-digested peptides. Furthermore, for the B1e13 and B14e21 peptides selected for quantification, we observed a >41-fold enhancement in ESI response. Because the derivatized peptides exhibited enhanced protonation and decreased polarity associated with the amino-group and carboxyl-group modification, the peptides were capable of being detected with higher sensitivity due to the improved ionization response from ESI. Validation study Table 2 shows the coefficients of determination for the calibration curves, the intraday precision values of the peak areas, and the LODs for the derivatized peptides. As a calibrant solution of human insulin, the stock solution of human insulin was diluted with 0.1% (v/v) BSA. The calibration curves of each peptide were calculated from the peak-area ratio of natural human insulin to human insulin-D40. The calibration curves showed good linearity, and the correlation coefficients were >0.9987 for all examined concentration ranges. The obtained calibration curves described in Fig. 3. The relative standard deviations of the peak areas obtained from the intraday determinations (n ¼ 6) of standard solutions were within 4.3%, and the LODs of the peptides were 17 pmol/L and 6.2 pmol/L. Therefore, all examined peptides were detected with a high degree of sensitively and capable of being quantified using the method presented here. Furthermore, the recoveries of B1e13- and B14e21 peptides from 10 ng/mL spiked serum were 100.4 ± 1.7% and 100.3 ± 1.7%, respectively, whereas they were 106.6 ± 6.8% and 101.4 ± 2.2%, respectively, from 1 ng/mL spiked serum. The result of obtaining these good recoveries show that it was able to cancel experimental procedures and MS detection errors by IDMS using insulin-D40 and to measure accurate human serum insulin. These results demonstrated that the method presented here enabled accurate analysis and quantification of human insulin in serum samples. Quantification of peptides in serum The human insulin concentration of the five serum samples

Fig. 3. Calibration curves of human insulin in the range of 0.05e10 ng/mL.

obtained from analysis using the method presented in this study and the precision of the MS/MS measurements are shown in Table 3. These human insulin concentrations were measured at 1.07 ng/mL, 0.29 ng/mL, 0.70 ng/mL, 1.79 ng/mL, and 1.23 ng/mL according to chemiluminescence enzyme immunoassay results. Comparison of measurements obtained using both methods indicated good linear correlation (r2 ¼ 0.9943 and 0.9944, respectively) between the results, suggesting a strong correlation between the IDMS-based method and the commercial immunochemical methods for assessing serum human-insulin concentrations. The obtained correlation curves described in Fig. 4. Values associated with the chemiluminescence enzyme immunoassay were higher as compared with those obtained using the method presented in this study. IDMS is able to determine the concentration of intact peptides in human insulin, whereas commercial immunoassays measure concentrations in human insulin based on immunoreactivity. Therefore, the overestimation of concentration by the commercial assay might be a consequence of cross-reactivity with incomplete cleavage products of human insulin. A highly specific IDMS-based

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Table 3 Quantification values obtained using the method presented in this study. Digested peptide

Lot 1 serum sample

Lot 2 serum sample

Lot 3 serum sample

Lot 4 serum sample

Lot 5 serum sample

B1e13 peptide B14e21 peptide

0.99 ± 0.025 0.95 ± 0.021

0.21 ± 0.012 0.20 ± 0.0089

0.62 ± 0.019 0.64 ± 0.016

1.5 ± 0.032 1.5 ± 0.030

1.1 ± 0.026 1.1 ± 0.024

Average

0.97 ± 0.019

0.21 ± 0.0046

0.63 ± 0.012

1.5 ± 0.016

1.1 ± 0.020

spiked serum were 100.4 ± 1.7% and 100.3 ± 1.7%, respectively, whereas they were 106.6 ± 6.8% and 101.4 ± 2.2%, respectively, from 1 ng/mL spiked serum. The result of obtaining these good recoveries show that it was able to cancel experimental procedures and MS detection errors by IDMS using insulin-D40 and to measure accurate human serum insulin. Furthermore, comparison of this method with chemiluminescence enzyme immunoassay revealed highly correlated insulin quantification results for serum concentrations of between 0.20 ng/mL and 1.51 ng/mL. These findings suggested that this method enabled traceable measurement of insulin from human serum, and represents a reference method that can be potentially used to standardize serum human insulin measurement and/or applied for the accurate determination or relative quantification of other peptides or proteins in biological fluids. Acknowledgments We acknowledge to Ms. Mariko Yoshioka and Ms. Ryoko Mizuno for technical assistance. Funding This study was supported by a Grant-in-Aid for Young Scientists (B) (Grant No. 63018624) from the Japan Society for the Promotion of Science. Conflict of interest The authors declare that they have no conflicts of interest regarding the contents of this article. References

Fig. 4. Correlation curves between the IDMS-based method and the commercial immunochemical methods for assessing serum human-insulin concentrations.

quantification method can eliminate such potential cross-reactivity and enable a reference-measurement procedure for standardized studies of serum human insulin. Conclusions In this study, we described the development and assessment of a sensitive and accurate analytical method for analyzing human insulin in serum samples via IDMS. Six digested peptides obtained from human insulin were successfully derivatized with PP, resulting in a 23- (A5e17 peptide) to 49-fold (B14e21 peptide) enhancement of sensitivity using these analytical techniques. This derivatization method was capable of being combined with other enrichment methods, such as SPE and immunoaffinity purification, to further increase sensitivity (sample enrichment, followed by derivatization). The recoveries of B1e13- and B14e21 peptides from 10 ng/mL

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