A simple and sensitive assay for eflornithine quantification in rat brain using pre‐column derivatization and UPLC‐MS/MS detection

Eflornithine (α‐difluoromethylornithine) has been used to treat second‐stage (or meningoencephalitic‐stage) human African trypanosomiasis and currently is under clinical development for cancer prevention. In this study, a new ultraperformance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS)‐based assay was developed and validated for the quantification of eflornithine in rat brain. To improve chromatographic retention and MS detection, eflornithine was derivatized with 6‐aminoquinolyl‐N‐hydroxysuccinimidyl carbamate for 5 min at room temperature prior to injection. Derivatized eflornithine was separated on a reverse‐phase C₁₈ UPLC column with a 6‐min gradient; elution occurred at approximately 1.5 min. Prior to derivatization, eflornithine was reproducibly extracted from rat brain homogenate by methanol protein precipitation (~70% recovery). Derivatized eflornithine was stable in the autosampler (6 °C) for at least 24 h. This new assay had acceptable intra‐ and interday accuracy and precision over a wide dynamic range (5000‐fold) and excellent sensitivity with a lower limit of quantification of 0.1 µm (18 ng/mL) using only 10 μL of rat brain homogenate. The validated eflornithine assay was applied successfully to determine eflornithine distribution in different regions of rat brain in an in situ rat brain perfusion study. Copyright © 2014 John Wiley & Sons, Ltd.     

Solid‐phase extraction and residue determination of glyphosate in apple by ion‐pairing reverse‐phase liquid chromatography with pre‐column derivatization

A new method for glyphosate residue determination in apple has been developed. A SPE cartridge was used to clean up the samples before derivatization. Glyphosate was derivatized with 4‐chloro‐3,5‐dinitrobenzotrifluoride (CNBF) and quantified by reverse ion‐pair liquid chromatography using cetyltrimethylammonium bromide (CTAB) as ion‐pair reagent. In pH 9.5 H₃BO₃–Na₂B₄O₇ medium, the reaction of glyphosate with CNBF was complete after 30 min at 60°C. The stability of the derivative on exposure to light at room temperature in methanol–water was demonstrated. The labeled glyphosate was separated on a Kromasil C₁₈ column (250×4.6 mm, 5 μm) at room temperature and UV detection was applied at 360 nm. Separation was achieved within 15 min in gradient elution mode. The correlation coefficient for the method was 0.9998 at concentrations ranging from 0.1 to 50 μg/g. The calculated recoveries for glyphosate in apple were from 86.00 to 99.55%, and the relative standard deviations (n = 6) were from 1.43 to 6.32. The limit of detection was 0.01 μg/g for glyphosate in apple.     

Determination of rimantadine in human urine by HPLC using a monolithic stationary phase and on‐line post‐column derivatization

In the present study, we propose the first HPLC method coupled to postcolumn derivatization for the determination of rimantadine in human urine samples. The analyte and amantadine (internal standard) were isocratically separated using an RP monolithic stationary phase (100 × 4.6 mm id) with a mobile phase consisting of CH₃OH/phosphate buffer (25 mmol/L, pH 3.0) at a volume ratio of 50:50. Postcolumn derivatization involved on‐line reaction with o‐phthalaldehyde (20 mmol/L) and N‐acetyl‐cysteine (5 mmol/L) at alkaline medium (100 mmol/L borate pH 11.0). Spectrofluorimetric detection at λ_ex/λ_em = 340/455 nm enabled the selective and sensitive determination of rimantadine in urine samples at a range of 50–500 ng/mL with an LOD of 5 ng/mL. Human urine samples were analyzed successfully after SPE using hydrophilic‐lipophilic balanced RP cartridges (30 mg/mL, Oasis HLB). Recoveries ranged between 89.7 and 102.7%.     

The stable o‐phthalaldehyde‐ferrocene/6‐ferrocenyl‐1‐hexanethiol pre‐column derivatization high‐performance liquid chromatography of dimethylarginine by novel dual fluorescence and electrochemical detector

Monomethylarginine, asymmetric dimethylarginine and symmetric dimethylarginine were separated on a Wakopak Combi ODS with an acetonitrile–100 mm potassium phosphate buffer (pH 7.0; 1:1, v/v). Dimethylarginines were derived from o‐phthalaldehyde for the fluorescence detector and from 6‐ferrocenyl‐1‐hexanethiol for the electrochemical detector. The detection limits of the dimethylarginines in spiked plasma were 0.3–0.5 pmol by electrochemical detection and 1–2 pmol by fluorescence detection. The detection limits were improved over 30 times by electrochemical detection and 10 times by fluorescence detection compared with previous reports. In previous derivatization liquid chromatography, the reaction solutions, o‐phthalaldehyde, 2‐mercaptethanol and dimethylarginines were unstable and required quick derivatization at 4°C. By our proposed pre‐column methods, the dimethylarginines were derivatized at room temperature and the fluorescent products were stable for 6 h. The manipulation performance was greatly advanced compared with previous LC reports. This is the first report on stable and sensitive dimethylarginines by dual detection. The selectivity was also improved by dual detection. The proposed method was applied to preliminary monitoring of dimethylargines in plasma and urine. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of acrolein in serum by high‐performance liquid chromatography with fluorescence detection after pre‐column fluorogenic derivatization using 1,2‐diamino‐4,5‐dimethoxybenzene

Acrolein is a major unsaturated aldehyde that is generated during the lipid peroxidation process. The measurement of acrolein in biological samples should be useful to estimate the degree of lipid peroxidation and to evaluate the effect of hazardous properties of acrolein on human health. In this study, a highly sensitive and selective high‐performance liquid chromatography with fluorescence detection method was developed for the determination of acrolein in human serum. The proposed method involves the pre‐column fluorogenic derivatization of acrolein with 1,2‐diamino‐4,5‐dimethoxybenzene (DDB) as a reagent. The fluorescent derivative of acrolein could be detected clearly without any interfering reagent blank peaks because DDB does not have intrinsic fluorescence itself, and the detection limit was 10 nM (signal‐to‐noise ratio = 3). The proposed method could selectively detect acrolein in human serum with a simple protein precipitation treatment. Copyright © 2015 John Wiley & Sons, Ltd.     

Enantiomeric separation of d‐ and l‐serine using high‐performance liquid chromatography with electrochemical detection following pre‐column diastereomer derivatization

Enantiomeric separation of d ‐ and l ‐serine on an octadecylsilica column was investigated using (2R)‐2,5‐dioxopyrrolidin‐1‐yl‐2,5,7,8‐tetramethyl‐6‐(tetrahydro‐2H‐pyran‐2‐yloxy)chroman‐2‐carboxylate (R‐NPCA), which was developed for a pre‐column derivatization reagent for electrochemical detection. In addition, (2S)‐2,5‐dioxopyrrolidin‐1‐yl‐2,5,7,8‐tetramethyl‐6‐(tetrahydro‐2H‐pyran‐2‐yloxy)chroman‐2‐carboxylate (S‐NPCA) was newly synthesized from (S)‐(?)‐6‐hydroxy‐2,5,7,8‐tetramethylchroman‐2‐carboxylic acid (S‐α‐CA), and the enantiomeric separation of d ‐ and l ‐serine using S‐NPCA was also examined. The enantiomeric separation of d ,l ‐serine was achieved using the R‐ or S‐NPCA as a chiral derivatization reagent, and the elution orders of the enantiomers were reversed between R‐ and S‐NPCA. The elution orders of d ‐ and l ‐serine unexpectedly reversed between the phosphate buffer at pH 4.0 and pH 2.2, both of which were used in the mobile phase. Separation factors obtained using R‐ and S‐NPCA were similar—1.09 and 1.07, respectively. The detection limit was approximately 940 fmol on the column (signal‐to‐noise ratio 3) when the applied voltage was +650 mV. Copyright © 2009 John Wiley & Sons, Ltd.     

Quantification of bisphenol A and its selected analogs in serum using pre‐column derivatization with high‐performance liquid chromatography and tandem mass spectrometry

Due to regulation of the use of bisphenol A, several analogs serving as bisphenol A replacements have drawn substantial attention for their adverse health effects. To investigate their occurrence in humans and identify possible pollution sources, it is necessary to develop a sensitive method for total bisphenols detection. Thus, a method based on enzymolysis and liquid‐liquid extraction followed by molecularly imprinted polymer solid‐phase extraction and pre‐column derivatization with high‐performance liquid chromatography and tandem mass spectrometry was proposed. The developed method exhibited superior selectivity and sensitivity. The matrix effect can be eliminated to a great extent. The method detection limits for eight bisphenols were 0.05～0.19 ng/mL. Satisfactory recoveries (71～119%) were obtained by spiking bovine serum at three levels (0.8, 8 and, 20 ng/mL). The method was successfully applied to determine total bisphenols in the serum samples of children. Bisphenol A, bisphenol F, bisphenol S, bisphenol B and bisphenol F were detected with concentrations from below the method detection limit to 1.65, 0.45, 0.79, 2.04 and 0.17 ng/mL, respectively. These results indicate that bisphenol A remains the major pollutant among the studied bisphenols in children, whereas threats from bisphenol A analogs should also be monitored.     

Determination of intracellular glutathione and cysteine using HPLC with a monolithic column after derivatization with monobromobimane

An optimized high‐performance liquid chromatography (HPLC) method is used to show that, as myoblasts differentiate into multinucleated muscle fibers, there is a shift to a more oxidized cell redox state. The HPLC method incorporated derivatization with monobromobimane for the determination of the reduced (GSH) and oxidized (GSSG) forms of glutathione and the reduced (Cys) and oxidized (CysSS) forms of cysteine. The derivatization was optimized to improve the sensitivity of the approach; the limits of detection for glutathione and cysteine were 3 × 10^?8 and 5 × 10^?8 M , respectively. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of total retronecine esters‐type hepatotoxic pyrrolizidine alkaloids in plant materials by pre‐column derivatization high‐performance liquid chromatography

A pre‐column derivatization high‐performance liquid chromatography method with diode array detection was developed and validated to determine the total retronecine esters‐type hepatotoxic pyrrolizidine alkaloids (RET‐HPAs) in herbs. The RET‐HPAs reacted with o‐chloranil in methanolic solution heated for 3 h, and an oxidative derivative was produced that could be detected at a maximal absorption of 223 nm. The analysis was performed using a C₁₈ column with an isocratic elution of methanol and aqueous 0.01% triethylamine (adjusted to pH 4 with formic acid), and the detection was carried out with DAD at 223 nm. The validation of the method included linearity, sensitivity, recovery and stability. It showed a good linear regression (r² > 0.9900) in the range of 2.5–250 µm with a limit of detection (S/N = 3) of 0.5 µm . The method provided desirable repeatability with overall intra‐ and inter‐day variations of less than 4.6%. The obtained recoveries for both of the extraction and derivatization process were between 94.6 and 100.7% (n = 3). Copyright © 2009 John Wiley & Sons, Ltd.     

Rapid determination of oxindole alkaloids in cat's claw by HPLC using ionic liquid‐based microwave‐assisted extraction and silica monolithic column

Cat's claw is a large woody vine with hook‐like thorns, and has been traditionally used to treat inflammatory disorders in South and Central America. In this study, a rapid, validated high‐performance liquid chromatographic (HPLC) method using a silica monolithic column was developed for the simultaneous determination of oxindole alkaloids, namely rhynchophylline, pteropodine, isomitraphylline and isopteropodine, in cat's claw. The ionic liquid‐based microwave‐assisted extraction (ILMAE), considered as an environmentally friendly and powerful tool, was first applied in the extraction of oxindole alkaloids. To optimize the HPLC method, the stationary phases, pH values of mobile phase and flow rates were investigated. The validated HPLC method using a Monolithic RP_18e column (100 × 4.6 mm) enables these analytes to be separated almost twice as fast as with a conventional particulate column (~16 vs ~30 min) with limits of quantification and detection of 0.5 and 0.15 μg/mL, respectively. The ILMAE conditions were optimized by the Taguchi orthogonal array design. In comparison with conventional water boiling extraction, ILMAE offers almost four times higher yields within an extremely short extraction time. The developed HPLC coupled with ILMAE method could be efficient and practical for rapid determination of oxindole alkaloids in cat's claw.     

Development and validation of a high‐performance liquid chromatography method for determination of lisinopril in human plasma by magnetic solid‐phase extraction and pre‐column derivatization

A sensitive, reliable and simple HPLC method was developed for the determination of lisinopril in human plasma. The method consists of extraction and clean‐up steps based on magnetic solid‐phase extraction and pre‐column derivatization with a fluorescent reagent. The mobile phase consisted of a mixture of methanol–sodium dihydrogen phosphate (pH 3.0; 0.005 m ; 75:25, v/v). The flow rate was set at 0.7 mL/min. Fluorescence detection was performed at 470nm excitation and 530nm emission wavelengths. Total chromatography run time was 5 min. The average extraction recovery of lisinopril and fluvoxamine (internal standard) was ≥82.8%. The limits of detection and quantification were determined as 1 and 3 ng/mL respectively. The method exhibited a linear calibration line over the concentration range of 3–1000 ng/mL with coefficient of determination (r²) of ≥0.98. The within‐run and between‐run precisions were satisfactory with values of CV of 1.8–12.8% (accuracy from 99.2 to 94.7%) and 2.4–13.7% (accuracy from 99.5 to 92.2%), respectively. These developments led to considerable improvement in method sensitivity and reliability. The method was validated according to the US Food and Drug Administration guidelines. Therefore, it can be considered as a suitable method for determination of lisinopril in plasma samples.     

On.column pre‐concentration of alcohol dehydrogenase in capillary electrophoresis

The analysis of alcohol dehydrogenase (ADH) at low concentration using capillary electrophoresis is described. Several simple and effective ways to improve detection limits and sensitivity are investigated. These include large volume sample stacking, head column field amplified sample stacking, and sweeping. Results indicate that by using a combination of head‐column field amplified sample stacking and sweeping, fluorescently labelled alcohol dehydrogenase can be pre‐concentrated online by dissolving samples in water or other low conductivity matrices, and injecting into a high conductivity micellar buffer. The abrupt changes in conductivity cause narrowing of the analyte length and thus enhance the detection sensitivity. Combination of this approach with laser induced fluorescence detection yields a limit of detection of 5×10^–13 M. Both qualitative and quantitative aspects of this method are investigated.     

Fully automated reagent peak‐free liquid chromatography fluorescence analysis of highly polar carboxylic acids using a column‐switching system and fluorous scavenging derivatization

In this study, we combined a column‐switching system with a fluorous scavenging derivatization method to develop a fully automated reagent peak‐free LC fluorescence detection protocol for the analysis of highly polar carboxylic acids. In this method, highly polar carboxylic acids were derivatized with fluorescent 1‐pyrenemethylamine in the presence of 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide and 1‐hydroxy‐1H‐benzotriazole. Residual excess of the unreacted reagent was tagged with 2‐(perfluorooctyl)ethyl isocyanate and then removed selectively using a fluorous column‐switching system placed in front of an analytical reversed‐phase column. The signal of the fluorous‐tagged unreacted reagent was completely absent in the resulting chromatograms; therefore, it did not interfere with the quantification of each acid especially those eluted before 20 min. The detection limits (S/N = 3) for the examined acids were in the range from 4.0 to 22 fmol per injection. We have applied this method to comparative analysis of highly polar carboxylic acids in urine samples obtained from diabetes mellitus type‐II model mice and their control.     

Synthesis of benzofurazan derivatization reagents for short chain carboxylic acids in liquid chromatography/electrospray ionization‐tandem mass spectrometry (LC/ESI‐MS/MS)

Benzofurazan derivatization reagents, 4‐[2‐(N,N‐dimethylamino)ethylaminosulfonyl]‐7‐(2‐aminopentylamino)‐2,1,3‐benzoxadiazole (DAABD‐AP) and 4‐[2‐(N,N‐dimethylamino) ethylaminosulfonyl]‐7‐(2‐aminobutylamino)‐2,1,3‐benzoxadiazole (DAABD‐AB), for short‐chain carboxylic acids in liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) were synthesized. These reagents reacted with short chain carboxylic acids in the presence of the condensation reagents at 60°C for 60 min. The generated derivatives were separated on the reversed‐phase column and detected by ESI‐MS/MS with the detection limits of 0.1–0.12 pmol on column. Upon collision‐induced dissociation, a single and intense product ion at m/z 151 was observed. These results indicated that DAABD‐AP and DAABD‐AB are suitable as the derivatization reagents in LC/ESI‐MS/MS analysis. Copyright © 2008 John Wiley & Sons, Ltd.     

Rapid determination of polycyclic aromatic hydrocarbons in rainwater by liquid–liquid microextraction and LC with core‐shell particles column and fluorescence detection

Liquid–liquid microextraction coupled to LC with fluorescence detection for the determination of Environmental Protection Agency's 16 priority pollutant polycyclic aromatic hydrocarbons in rainwater has been developed. The optimization of the extraction method has involved several parameters, including the comparison between an ultrasonic bath and a magnetic stirrer as extractant apparatus, the choice of the extractant solvent, and the optimization of the extraction time. Liquid–liquid microextraction gave good results in terms of recoveries (from 73.6 to 102.8% in rainwater) and repeatability, with a very simple procedure and low solvent consumption. The reported chromatographic method uses a Core‐Shell technology column, with particle size <3 μm instead of classical 5‐μm particles column. The resulting backpressure was below 300 bar, allowing the use of a conventional HPLC system rather than the more expensive ultrahigh performance LC (UHPLC). An average decrease of 59% in run time and 75% in eluent consumption has been obtained, compared to classical HPLC methods, keeping good separation, sensitivity, and repeatability. The proposed conditions were successfully applied to the determinations of polycyclic aromatic hydrocarbons in genuine rainwater samples.     

Simultaneous determination of four aflatoxins and ochratoxin A in ginger and related products by HPLC with fluorescence detection after immunoaffinity column clean‐up and postcolumn photochemical derivatization

Ginger, a widely used spice and traditional Chinese medicine, is prone to be contaminated by mycotoxins. A simple, sensitive, and reproducible method based on immunoaffinity column clean‐up coupled with HPLC and on‐line postcolumn photochemical derivatization with fluorescence detection was developed for the simultaneous determination of aflatoxins (AFs) B₁, B₂, G₁, G₂, and ochratoxin A (OTA) in 25 batches of gingers and related products marketed in China for the first time. The samples were first extracted by ultrasonication with methanol/water (80:20, v/v) and then cleaned up with immunoaffinity columns for analysis. Under the optimized conditions, the LODs and LOQs for the five mycotoxins were 0.03–0.3 and 0.1–0.9 μg/kg, respectively. The average recoveries ranged from 81.3–100.8% for AFs and from 88.6–99.5% for OTA at three spiking levels. Good linearity was observed for the analytes with correlation coefficients all >0.9995. All moldy gingers were contaminated with at least one kind of the five investigated mycotoxins, while none of them were found in normal gingers. Ginger powder samples were contaminated slightly with the contamination levels below the LOQs, while ginger tea bags were mainly contaminated by OTA at 1.05–1.19 μg/kg and ginger black tea bags were mainly contaminated by AFs at 3.37–5.76 μg/kg. All the contamination levels were below the legally allowable limits.     

Automated derivatization and analysis of malondialdehyde using column switching sample preparation HPLC with fluorescence detection

Analyte derivatization is advantageous for the analysis of malondialdehyde (MDA) as a biomarker of oxidative stress in biological samples. Conventionally, however, derivatization is time consuming, error-prone and has limited options for automation. We have addressed these challenges for the solid phase analytical derivatization of MDA from small volume tissue homogenate samples. A manual derivatization method was first developed using Amberlite XAD-2 (12 mg) as the solid phase. Subsequently an automated column switching process was developed that provided simultaneous derivatization and extraction of the MDA-DH hydrazone product on a cartridge packed with XAD-2, followed by quantitative elution of the product to an analytical LC column (Waters NovoPak C18, 3.9 x 150 mm). The LOD was 0.02 microg/mL and recovery was quantitative. The method was linear (r(2) >0.999) with precision < 5% from the LOQ (0.06 microg/mL) to at least 35 microg/mL. The method was successfully applied to the analysis of small volume (30 microL) mouse tissue homogenate samples. Endogenous levels of MDA in the tissues ranged from 20 to 40 nmol/g tissue (ca. 0.1-0.2 microg/mL homogenate). Compared to conventional MDA analyses, the current method has advantages in automation, selectivity, precision and sensitivity for analysis from very small sample volumes.     

Quantification of peptides using N‐terminal isotope coding and C‐terminal derivatization for sensitive analysis by micro liquid chromatography‐tandem mass spectrometry

Stable isotope‐coding coupled with mass spectrometry is a popular method for quantitative proteomics and peptide quantification. However, the efficiency of the derivatization reaction at a particular functional group, especially in complex structures, can affect accuracy. Here, we present a dual functional‐group derivatization of bioactive peptides followed by micro liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). By separating the sensitivity‐enhancement and isotope‐coding derivatization reactions, suitable chemistries can be chosen. The peptide amino groups were reductively alkylated with acetaldehyde or acetaldehyde‐d₄ to afford N‐alkylated products with different masses. This process is simple, quick and high‐yield, and accurate comparative analysis can be achieved for the mass‐differentiated peptides. Then, the carboxyl groups were derivatized with 1‐(2‐pyrimidinyl)piperazine to increase MS/MS sensitivity. Angiotensins I–IV, bradykinin and neurotensin were analyzed after online solid phase extraction by micro LC‐MS/MS. In all instances, a greater than 17‐fold increase in sensitivity was achieved, compared with the analyses of the underivatized peptides. Furthermore, the values obtained from the present method were in agreement with the result from isotope dilution quantification using isotopically labeled angiotensin I [Asp‐Arg‐(Val‐d₈)‐Tyr‐Ile‐His‐Pro‐(Phe‐d₈)‐His‐Leu]. Copyright © 2016 John Wiley & Sons, Ltd.     

Determination of fluvoxamine in rat plasma by HPLC with pre-column derivatization and fluorescence detection using 4-fluoro-7-nitro-2,1,3-benzoxadiazole

A sensitive, simple and reliable method using high-performance liquid chromatographic (HPLC) assay of fluvoxamine (FLU), a selective serotonin reuptake inhibitor (SSRI), in rat plasma after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was developed in this study. Extracted plasma samples were mixed with NBD-F at 60 degrees C for 5 min and injected into HPLC. Retention times of FLU and an internal standard (propafenone) derivative were 15.5 and 13.5 min, respectively. The calibration curve was linear over the range 0.015-1.5 microg/mL (r2 = 0.9985) and the lower limits of detection and quantification of FLU were 0.008 and 0.015 microg/mL, respectively, in 100 microL of plasma. The derivative sample was stable at 4 degrees C for 1 day. The coefficients of variation for intra-day and inter-day assay of FLU were less than 8.3 and 9.6%, respectively. Other SSRIs and centrally acting drugs did not interfere with the peak of the FLU derivative. The method was applied for analysis of the plasma samples from rats treated with FLU. These results indicate that the method presented is useful to determine the FLU levels in rat plasma of volumes as small as 100 microL and can be applied to pharmacokinetic studies.