With or Without Internal Standard in HPLC Bioanalysis. A Case Study

Imre, Silvia; Tero-Vescan, Amelia; Dogaru, Maria Titica; Kelemen, László; Muntean, Daniela-Lucia; Curticăpean, Augustin; Szegedi, Noémi; Vari, Camil-Eugen

doi:10.1093/chromsci/bmy106

Cited by 17 publications

(11 citation statements)

References 24 publications

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“…This method correlates the ratio of the analyte response to the response of the IS with the ratio of the analyte amount to the IS amount. [ 83,85 ]…”

Section: Uncertaintymentioning

confidence: 99%

Experimental methods in chemical engineering: High performance liquid chromatography—HPLC

et al. 2021

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High performance liquid chromatography (HPLC) identifies compounds in a sample and quantifies their concentration. The instrument consists of an injector that introduces a μL sample to a mobile phase that carries the analytes across a column filled with an adsorbing substance. The analytes adsorb and desorb at different rates as they flow down the column and are thereby separated. Detectors record a signal proportional to the concentration of the analyte in the mobile phase. Laboratories apply modern HPLC systems for research and development, quality control, safety, and validation. Academia and industry resort to HPLC to purify, identify, and characterize a wide variety of molecules at all stages of a process. Developing an HPLC analytical method is a long and laborious process requiring standards, calibration curves, derivatization, and multiple tests to identify an appropriate column, stationary phase, flow rate, temperature, and mobile phase. Optimal operating conditions maintain a good resolution (high signal‐to‐noise ratio and low peak overlap) while minimizing analysis time. Web of Science Core Collection indexed 8900 articles in 2019, which is relatively few compared to x‐ray and scanning electron microscopy. However, it is such an ubiquitous technique and we estimate that the number of articles that report it in the experimental section is an order of magnitude higher. The bibliometric analysis of the keywords identified four research clusters: phenolics and flavonoids, extraction and pharmacokinetics, in vitro and oxidative stress, and optimization. Here, we detail a walkthrough for the basic steps required to develop a chromatographic method.

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“…This method correlates the ratio of the analyte response to the response of the IS with the ratio of the analyte amount to the IS amount. [ 83,85 ]…”

Section: Uncertaintymentioning

confidence: 99%

Experimental methods in chemical engineering: High performance liquid chromatography—HPLC

et al. 2021

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“…The association constant is also of the order of 10 5 M -1 [4,21]. The choice of MXC -PXC combination was made based on the fact that often the two substances are used in tandem analyte -internal standard for quantification from biological samples using chromatographic techniques [14]. In the present study, piroxicam was used as an internal standard (ISTD).…”

Section: Figurementioning

confidence: 99%

“…The conditions for the chromatographic analysis were established after a slight modification of a previously published method by Imre S et al [14]: the same HPLC system Agilent Technologies 1100 Series type was used and the analytical column was Zorbax SB-C18 (Solvent Saver Plus 100 x 3 mm, 3.5 µm). The column temperature was set at 45°C.…”

Section: Chromatographic Conditionsmentioning

confidence: 99%

Evaluating the Capacity of Human Serum Albumin to Reduce Non-Specific Binding of Meloxicam in the Ultrafiltration Process

IMRE¹

2021

FARMACIA

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Due to the importance that plasma protein binding of drugs plays and the constant interest to optimize study methods of the process, this study aimed to test the capacity of a human serum albumin solution to reduce non-specific binding of highly bound meloxicam, based on the ability of the protein to block most non-specific binding sites of the ultrafiltration devices. Samples of meloxicam and internal standard piroxicam were prepared in both phosphate-buffered saline solution, commonly used in biological research, and 5% human serum albumin solution. After the ultrafiltration process, the free drug fraction was analysed using a reversed phase HPLC-UV method. A decrease in the concentration of meloxicam after ultrafiltration in both matrices was observed, the decrease being higher in the case of phosphate-buffered saline solution. The mean determined degree of binding to albumin for meloxicam was 55%. The high decrease in concentration for meloxicam in the non-proteic matrix indicates a very high degree of non-specific binding, phenomenon which appears to be indeed reduced for the samples prepared in human serum albumin solution. The study also emphasises the very close attention that should be paid to the preparation and processing of samples in protein matrices and to the experimental conditions. RezumatDatorită importanței pe care legarea medicamentelor de proteinele plasmatice o prezintă și a interesului constant de a optimiza metodele de studiu ale procesului, acest studiu a avut ca scop testarea capacității unei soluții de albumină serică umană de a reduce legarea nespecifică a meloxicamului puternic legat, pe baza capacității proteinei de a bloca majoritatea situsurilor de legare nespecifică a dispozitivelor de ultrafiltrare. Probele de meloxicam și standard intern piroxicam au fost preparate atât în soluție de tampon fosfat salin, utilizată în mod obișnuit în cercetări biologice, cât și în soluție de albumină serică umană 5%. După procesul de ultrafiltrare, fracția de medicament liber a fost analizată folosind o metodă HPLC-UV în fază inversă. S-a observat o scădere a concentrației de meloxicam după ultrafiltrare în ambele matrici, scăderea fiind mai mare în cazul soluției de tampon fosfat salin. Gradul mediu de legare de albumină determinat pentru meloxicam a fost de 55%. Scăderea ridicată a concentrației de meloxicam în matricea non-proteică indică un grad foarte ridicat de legare nespecifică, fenomen care pare a fi într-adevăr redus pentru probele preparate în soluție de albumină serică umană. Studiul scoate în evidență, de asemenea, atenția mare care trebuie acordată condițiilor experimentale, preparării și prelucrării probelor în matrici proteice.

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“…Three assays described in this review used an ExS to correct for the variations (Buchner et al, ; Dhananjeyan et al, ; Guichard et al, ). Although it is well known that ISs reduce the effect of interfering matrix components, minimizes sample processing errors and the variability of detection, ISs are not always required (Imre et al, ). ISs standards are preferred due to being more accurate.…”

Section: Internal Standardsmentioning

confidence: 99%

A review of the bioanalytical methods for the quantitative determination of capecitabine and its metabolites in biological matrices

Knikman

Rosing

Guchelaar

et al. 2020

Biomedical Chromatography

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The bioanalysis of the oral anticancer drug capecitabine and its metabolites has been investigated extensively over the past years. This paper reviews methods for the bioanalysis of capecitabine and its metabolites. The focus of this review will be on sample pre‐treatment, chromatography and detection. Furthermore, the choice of standards and analytical problems encountered during analysis of capecitabine and its metabolites in biological matrices will be discussed. The major challenges in the bioanalysis of capecitabine and its metabolites are the simultaneous extraction and analysis due to the differences in polarity of the analytes. Furthermore we evaluate currently described methods for the quantification of capecitabine and its metabolites. Future wishes and perspectives are stated that could serve as an inspiration for further development of assays for the quantification of capecitabine and its metabolites.

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With or Without Internal Standard in HPLC Bioanalysis. A Case Study

Cited by 17 publications

References 24 publications

Experimental methods in chemical engineering: High performance liquid chromatography—HPLC

Experimental methods in chemical engineering: High performance liquid chromatography—HPLC

Evaluating the Capacity of Human Serum Albumin to Reduce Non-Specific Binding of Meloxicam in the Ultrafiltration Process

A review of the bioanalytical methods for the quantitative determination of capecitabine and its metabolites in biological matrices

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