Quantification of recombinant human erythropoietin by amino acid analysis using isotope dilution liquid chromatography–tandem mass spectrometry

Herein, we describe an accurate method for protein quantification based on conventional acid hydrolysis and an isotope dilution-ultra performance liquid chromatography–tandem mass spectrometry method. The analyte protein, recombinant human erythropoietin (rhEPO), was effectively hydrolyzed by incubation with 8 mol/L hydrochloric acid at 130 °C for 48 h, in which at least 1 μmol/kg of rhEPO was treated to avoid possible degradation of released amino acids during hydrolysis. Prior to hydrolysis, sample solution was subjected to ultrafiltration to eliminate potential interfering substances. In a reversed-phase column, the analytes (phenylalanine, proline, and valine) were separated within 3 min using gradient elution comprising 20 % (v/v) acetonitrile and 10 mmol/L ammonium acetate, both containing 0.3 % (v/v) trifluoroacetic acid. The optimized hydrolysis and analytical conditions in our study were strictly validated in terms of accuracy and precision, and were suitable for the accurate quantification of rhEPO. Certified rhEPO was analyzed using a conventional biochemical assay kit as an additional working calibrant for the quantification of EPO and improved the accuracy. The optimized protocol is suitable for the accurate quantification of rhEPO and satisfactorily serves as a reference analytical procedure for the certification of rhEPO and similar proteins.

Sensitive detection of enteropathogenic E. coli using a bfpA gene-based electrochemical sensor

We have developed a sensitive assay for enteropathogenic E. coli (EPEC) by integrating DNA extraction, specific polymerase chain reaction (PCR) and DNA detection using an electrode modified with the bundle-forming pilus (bfpA) structural gene. The PCR amplified products are captured on the electrode and hybridized with biotinylated detection probes to form a sandwich hybrid containing two biotinylated detection probes. The sandwich hybridization structure significantly combined the numerous streptavidin alkaline phosphatase on the electrode by biotin-streptavidin connectors. Electrochemical readout is based on dual signal amplification by both the sandwich hybridization structure and the enzyme. The electrode can satisfactorily discriminate complementary and mismatched oligonucleotides. Under optimal conditions, synthetic target DNA can be detected in the 1 pM to 10 nM concentration range, with a detection limit of 0.3 pM. EPEC can be quantified in the 10 to 10⁷ CFU mL^?1 levels within 3.5 h. The method also is believed to present a powerful platform for the screening of pathogenic microorganisms in clinical diagnostics, food safety and environmental monitoring.

High-throughput absolute quantification of proteins using an improved two-dimensional reversed-phase separation and quantification concatemer (QconCAT) approach

Stable isotope dilution–selective reaction monitoring–mass spectrometry (SID-SRM-MS) has been widely used for the absolute quantitative analysis of proteins. However, when performing the large-scale absolute quantification of proteins from a more complex tissue sample, such as mouse liver, in addition to a high-throughput approach for the preparation and calibration of large amounts of stable-isotope-labelled internal standards, a more powerful separation method prior to SRM analysis is also urgently needed. To address these challenges, a high-throughput absolute quantification strategy based on an improved two-dimensional reversed-phase (2D RP) separation and quantification concatemer (QconCAT) approach is presented in this study. This strategy can be used to perform the simultaneous quantification of hundreds of proteins from mouse liver within one week of total MS measurement time. By using calibrated synthesised peptides from the protein glutathione S-transferase (GST), large amounts of GST-tagged QconCAT internal standards corresponding to hundreds of proteins can be accurately and rapidly quantified. Additionally, using an improved 2D RP separation method, a mixture containing a digested sample and QconCAT standards can be efficiently separated and absolutely quantified. When a maximum gradient of 72 min is employed in the first LC dimension, resulting in 72 fractions, identification and absolute quantification experiments for all fractions can be completed within one week of total MS measurement time. The quantification approach developed here can further extend the dynamic range and increase the analytical sensitivity of SRM analysis of complex tissue samples, thereby helping to increase the coverage of absolute quantification in a whole proteome.

Immobilized purine nucleoside phosphorylase from Schistosoma mansoni for specific inhibition studies

The parasite Schistosoma mansoni (Sm) depends exclusively on the salvage pathway for its purine requirements. The enzyme purine nucleoside phosphorylase (PNP) is, therefore, a promising target for development of antischistosomal agents and an assay for screening of inhibitors. To enable this, immobilized SmPNP reactors were produced. By quantification of hypoxanthine by liquid chromatography, kinetic constants (K _M) for the substrate inosine were determined for the free and immobilized enzyme as 110 ± 6.90 μmol?L ^?1 and 164 ± 13.4 μmol?L ^?1 , respectively, indicating that immobilization did not affect enzyme activity. Furthermore, the enzyme retained 25 % of its activity after four months. Non-Michaelis kinetics for the phosphate substrate, and capacity for P_i-independent hydrolysis were also demonstrated, despite the low rate of enzymatic catalysis. Use of an SmPNP immobilized enzyme reactor (IMER) for inhibitor-screening assays was demonstrated with a small library of 9-deazaguanine analogues. The method had high selectivity and specificity compared with screening by use of the free enzyme by the Kalckar method, and furnished results without the need for verification of the absence of false positives.

Simultaneous serum desalting and total protein determination by macroporous reversed-phase chromatography

Macroporous reversed-phase (mRP) chromatography was successfully used to develop an accurate and precise method for total protein in serum. The limits of detection (0.83 μg, LOD) and quantification (2.51 μg, LOQ) for the mRP method are comparable with those of the widely used micro BCA protein assay. The mRP method can be used to determine the total protein concentration across a wide dynamic range by detecting chromatographic peaks at 215 nm and 280 nm. The method has the added advantage of desalting and denaturing proteins, leading to more complete digestion by trypsin and to better LC–MS–MS identification in shotgun proteomics experiments.

A multiresidual method based on ion-exchange chromatography with conductivity detection for the determination of biogenic amines in food and beverages

In the present work a sensitive and accurate method by ion chromatography and conductimetric detection has been developed for the determination of biogenic amines in food samples at microgram per kilogram levels. The optimized extraction procedure of trimethylamine, triethylamine, putrescine, cadaverine, histamine, agmatine, spermidine, and spermine from real samples, as well as the separation conditions based on a multilinear gradient elution with methanesulfonic acid and the use of a weak ionic exchange column, have provided excellent results in terms of resolution and separation efficiency. Extended calibration curves (up to 200 mg/kg, r?>?0.9995) were obtained for all the analyzed compounds. The method gave detection limits in the range 23–65 μg/kg and quantification limits in spiked blank real samples in the range 65–198 μg/kg. Recovery values ranged from 82 to 103 %, and for all amines, a good repeatability was obtained with precision levels in the range 0.03–0.32 % (n?=?4). The feasibility and potential of the method were tested by the analysis of real samples, such as tinned tuna fish, anchovies, cheese, wine, olives, and salami.

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.

Comparison of an HPTLC method with the Reflectoquant assay for rapid determination of 5-hydroxymethylfurfural in honey

5-Hydroxymethylfurfural (HMF) was analyzed in 17 botanical varieties of honey from 12 countries. A recently developed high-performance thin-layer chromatographic (HPTLC) method was limited because of increased matrix effects at higher honey sample loading. Therefore, the method was modified to achieve higher sensitivity and eliminate matrix interference by use of rectangular application combined with a focusing step. The HPTLC results were compared with results from the new spectrophotometric Reflectoquant hydroxymethylfurfural assay. Both methods had quantification limits of 4 mg kg^?1 and were suitable for rapid quantification of HMF in honey at the strictest regulated level of 15 mg kg^?1. Comparable results were obtained for the 17 honey samples, with a mean deviation of 2.9 mg kg^?1 (15 %). The optimized HPTLC method was proved to be highly matrix-robust and was validated for the 17 different honey matrices (correlation coefficients ≥0.9994 (n?=?6), mean intra-day precision 3.2 % (n?=?3 within a plate; n?=?2 repeated within a day), mean inter-day precision 3.7 % (n?=?3), mean reproducibility over the whole procedure including sample preparation 4.1 % (n?=?2), and mean recovery 106.9 % (n?=?5 different concentrations; n?=?4 different honey matrices). Recovery for a range of different application volumes, and thus for different honey matrix loading, differed by only ≤4.2 %. HMF results when calculated by use of external calibration and by use of the standard addition method varied by 8.8 %. Both revealed that any matrix effect was minor and that the original matrix interference problem was successfully solved.

Profiling thiol metabolites and quantification of cellular glutathione using FT-ICR-MS spectrometry

We describe preparation and use of the quaternary ammonium-based α-iodoacetamide QDE and its isotopologue *QDE as reagents for chemoselective derivatization of cellular thiols. Direct addition of the reagents to live cells followed by adduct extraction into n-butanol and analysis by FT-ICR-MS provided a registry of matched isotope peaks from which molecular formulae of thiol metabolites were derived. Acidification to pH 4 during cell lysis and adduct formation further improves the chemoselectivity for thiol derivatization. Examination of A549 human lung adenocarcinoma cells using this approach revealed cysteine, cysteinylglycine, glutathione, and homocysteine as principal thiol metabolites as well as the sulfinic acid hypotaurine. The method is also readily applied to quantify the thiol metabolites, as demonstrated here by the quantification of both glutathione and glutathione disulfide in A549 cells at concentrations of 34.4?±?11.5 and 10.1?±?4.0 nmol/mg protein, respectively.

Detection of mismatched caspase-3 DNA oligonucleotides with an SPR biosensor following amplification by Taq polymerase

We demonstrate that base mismatches of caspase-3 DNA sequences can be detected by surface plasmon resonance (SPR) following signal amplification by polymerase from Thermus aquaticus (Taq). The concentration of magnesium ions and the respective dNTPs for polymerase binding to the oligonucleotides on the sensing surface were optimized. Taq polymerase binds to double-stranded DNA that is self-assembled on the gold surface of the biosensor to induce an SPR signal. Experiments are presented on the effect of Mg(II) and dNTP concentrations on the activity of the polymerase on the sensing surface. The detection limits are 50 pM, 0.1 nM, 0.7 nM, 7 nM, and 20 nM for correctly matched, single-base mismatched, two-base mismatched, three-base mismatched and four-base mismatched DNA of caspase-3, respectively. This is attributed to the optimized experimental conditions, with samples containing 2 μM of Mg(II) and 0.3 mM of dNTP.

New validated HPLC methodology for the determination of (−)-trans-paroxetine and its enantiomer in pharmaceutical formulations with use of ovomucoid chiral stationary phase

A new chromatographic method for the enantioseparation and the determination of (?)-trans-paroxetine and (+)-trans-paroxetine has been developed with the aid of amylose ovomucoid-based chiral stationary phase. The method is faster and five times more sensitive than procedures recommended previously: limit of detection and limit of quantification are 5 and 16 ng/mL, respectively [modified (Ferretti et al. in J Chromatogr B 710:157–164, 1998): 20 and 60 ng/mL]. It was carefully validated and applied for the determination of (?)-trans-paroxetine and (+)-trans-paroxetine in Parogen (Mc Dermott Laboratories Ltd.) and Xetanor (Actavis) coated tablets.

Examination of an absolute quantity of less than a hundred nanograms of proteins by amino acid analysis

We developed an ultra-sensitive method of amino acid analysis (AAA) for the absolute quantification of less than 100 ng of proteins, in solution or on polyvinylidene difluoride (PVDF) membranes using an oxygen-free chamber for protein hydrolysis. We used a pre-label method with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate for fluorescence detection, ion-pair chromatography with a reversed-phase column, and an ultra-high-pressure high-performance liquid chromatography. We optimized both handling- and instrument-dependent factors for accurate quantification and showed that the least amount of proteins to quantify was determined by handling accuracy rather than instrumental limit for quantification which was 0.6 fmol/amino acid. As a new evaluation method for the handling accuracy, we adopted the protein identification by the obtained amino acid compositions by AAA and the Swiss-Prot database search without the restriction of species. As a result, the least amount of starting material for AAA was 16 ng (0.24 pmol) for a solution of bovine serum albumin (BSA), 33 ng (0.50 pmol) for BSA on a PVDF membrane, and 44 ng (0.15 pmol) for thyroglobulin on a PVDF membrane. These results demonstrate that the ultra-sensitive AAA developed in this study is feasible for absolute quantification of biological significant protein.

Development and validation of a microfluidic chip-based nano-liquid chromatography–triple quadrupole tandem mass spectrometry method for a sensitive and reliable quantification of 7-ethyl-10-hydroxycamptothecin (SN38) in mouse plasma

There is an increasing need for more sensitive analytical methods in pharmacokinetic studies, for example, for phase 0 clinical trials. A novel HPLC Chip–triple quadrupole mass spectrometer method (HPLC Chip-MS/MS method) for the quantification of 7-ethyl-10-hydroxycamptothecin (SN38) was developed, validated, and employed to the pharmacokinetic analysis of SN38 in ICR mice. Protein precipitation with a ratio of plasma/acetonitrile of 1:10 was chosen as the sample processing method. The nano-electrospray inserted in the microfluidic chip operated in positive mode, and selected reaction monitoring was used for quantification. Our bioanalytical method met all essential validation parameters—selectivity, accuracy, precision, dilution integrity, calibration curve, matrix effect, recovery, and different stability tests (benchtop, freeze–thaw, autosampler stability). The calibration curves (weight 1/x ²) were linear for the range 50–10,000 pg/mL. Clogging was not observed until the end of the lifetime of the microfluidic chip (350–400 injections), and carryover was practically eliminated through the introduction of a step gradient elution program. After intraperitoneal injection of 0.1 mg/kg irinotecan, SN38 concentration could be measured up to 6 h with accuracy and precision. Thus, we developed a new, very sensitive HPLC Chip-MS/MS method for the determination of plasma SN38 that has been validated in compliance with guidelines from different regulation authorities.

Electromagnetic induction-assisted heating as a new method for on-line cloud point extraction of cadmium from water samples

We report on a novel method for on-line cloud point extraction (CPE) for preconcentration of cadmium ions. It is based on electromagnetic induction-assisted heating (EMIH) of iron particles in a packed bed contained in a quartz tube that acts as an on-line CPE enrichment column. The cadmium complex of 1-(2-pyridylazo)-2-naphthol is quantitatively retained by the column under the cloud point temperature with the help of EMIH. The column was then eluted with alcoholic borax buffer at room temperature and on-line coupled to FAAS. Under optimum conditions, the limit of detection (3 s_b/b) and limit of quantification (10 s_b/b) are 0.21 μg?L^?1 and 0.70 μg?L^?1 of Cd(II), respectively, and the relative standard deviation is 3.8 % (for n?=?8; at 20 ng?mL^?1). An enhancement factor of 76 is typically achieved. The correlation coefficient of the calibration graph using the present method was 0.9986. The method was successfully applied to determine Cd(II) in water samples

Enantiomeric LC Separation of Epinephrine on Amylose based sorbent

A simple, rapid, and robust chiral HPLC method has been developed and validated for separation of the enantiomers of epinephrine, l-1-(3,4-dihydroxyphenyl)-2-(methylamino)ethanol, an antihypertensive drug, in the bulk drug. The enantiomers were resolved on an amylose-based stationary phase with n-hexane–2-propanol–methanol–trifluoroacetic acid–diethylamine 90:05:05:0.2:0.2 (v/v) as mobile phase at a flow rate of 1.0 mL min^?1. In the optimized method resolution between the enantiomers was not less than 3.0. The trifluoroacetic acid and diethylamine in the mobile phase were important for enhancing chromatographic efficiency and hence the resolution of the enantiomers. The method was extensively validated and proved to be robust. The calibration plot for the d enantiomer was highly linear over the concentration range 100–2,000 μg mL^?1. The limits of detection and quantification for the d enantiomer were 0.15 and 0.45 μg mL^?1, respectively. Recovery of the d enantiomer from bulk drug samples of epinephrine ranged between 99.5 and 101.5%. Epinephrine sample solution was stable for up to 48 h. The method was suitable for accurate quantitative determination of the d enantiomer in the bulk drug substance     

ATPase inhibitor based luciferase assay for prolonged and enhanced ATP pool measurement as an efficient fish freshness indicator

The nucleotide degradation pathway in somatic cells leads to the accumulation of products such as hypoxanthine and inosine, which are commonly used as fish and meat freshness indicators. Assays based on these molecules cannot differentiate the postmortem time over a short period of time (5–10 h). Further, quantification of these degradation products is cumbersome, costly and time-consuming. For the proposed assay, optimal concentrations of 30 and 2 mM, respectively, for the ATPase inhibitors sodium orthovanadate and EDTA were found. Further, it was observed that a firefly luciferase based assay could enhance the sensitivity levels up to 165-fold at 30 °C. In addition, it was observed that the sensitivity for ATP assay was enhanced up to 60-fold even after 12 h. The limit of detection for the ATP assay was 1 pM, unlike other conventional methods, which are sensitive only up to micromolar levels. Moreover, as little as 0.044 g fish fillet was required for the assay, and no time-consuming sample preparation was necessary. Luminescence of prolonged duration was observed in harvested fish kept at -20 °C in comparison with fish kept at 4 and 30 °C, which reflects the shelf life of fish preserved at lower temperatures.

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.

Characterization of liquid chromatography-tandem mass spectrometry method for the determination of acrylamide in complex environmental samples

This work describes the characterization of a solid-phase extraction (SPE) and liquid-chromatography-tandem mass spectrometry-based method for the analysis of acrylamide (AA) in complex environmental waters. The method involved the SPE of AA using activated carbon, and the AA was detected with tandem mass spectrometry after separating on an ion exclusion high-performance liquid chromatography column. The method incorporated two labeled AA standards for quantification using isotope dilution and to assess absolute extraction recovery. The method was evaluated for inter- and intra-day precision and accuracy. The method was both accurate (i.e., <30 % error) and precise (i.e., <20 % relative standard deviation), with absolute extraction recoveries averaging 37 %. The mass spectrometry provided excellent sensitivity, with instrumental limits of detection and quantitation values of 23 and 75 pg, respectively. The method detection limit was determined to be 0.021 μg/L. The analysis of AA was successfully performed in real-world samples that contained total dissolved solids concentrations ranging from 23,600 to 297,000 mg/L and AA concentrations ranging from 0.082 to 1.0 μg/L.

Partial enzymatic elimination and quantification of sarcosine from alanine using liquid chromatography–tandem mass spectrometry

Since sarcosine and d,l-alanine co-elute on reversed-phase high-performance liquid chromatography (HPLC) columns and the tandem mass spectrometer cannot differentiate them due to equivalent parent and fragment ions, derivatization is often required for analysis of sarcosine in LC/MS systems. This study offers an alternative to derivatization by employing partial elimination of sarcosine by enzymatic oxidation. The decrease in apparent concentration from the traditionally merged sarcosine–alanine peak associated with the enzymatic elimination has been shown to be proportional to the total sarcosine present (R ²?=?0.9999), allowing for determinations of urinary sarcosine. Sarcosine oxidase was shown to eliminate only sarcosine in the presence of d,l-alanine, and was consequently used as the selective enzyme. This newly developed technique has a method detection limit of 1 μg/L (parts per billion) with a linear range of 3 ppb–1 mg/L (parts per million) in urine matrices. The method was further validated through spiked recoveries of real urine samples, as well as the analysis of 35 real urine samples. The average recoveries for low, middle, and high sarcosine concentration spikes were 111.7, 90.8, and 90.1 %, respectively. In conclusion, this simple enzymatic approach coupled with HPLC/MS/MS is able to resolve sarcosine from d,l-alanine leading to underivatized quantification of sarcosine.

Single-drop microextraction for the determination of manganese in seafood and water samples

We describe a method for single drop microextraction of manganese from fish, mollusk, and from natural waters using the reagent 1-(2-pyridylazo)-2-naphthol as the complexing agent and chloroform as the fluid extractor. After extraction, the analyte was directly submitted to graphite furnace electrothermal atomic absorption spectrometry. Once optimized, the method has a detection limit of 30 ng L^?1, a limit of quantification of 100 ng L^?1, and an enrichment factor of 16. Its accuracy was verified by applying the procedure to the following certified reference materials: apple leaves, spinach leaves, bovine liver, and mussel tissue. The procedure was also successfully applied to the determination of manganese in seafood and natural waters.