A rapid LC/MS/MS quantitation assay for naringin and its two metabolites in rats plasma

doi:10.1016/j.jpba.2005.07.031

Journal of Pharmaceutical and Biomedical Analysis

Volume 40, Issue 2, 13 February 2006, Pages 454-459

https://doi.org/10.1016/j.jpba.2005.07.031 Get rights and content

Abstract

Naringin is a flavonoid that exists in many plants and traditional Chinese medicines. In this study, a highly sensitive and specific electrospray ionization (ESI) liquid chromatography–tandem mass spectrometry (LC/MS/MS) method was developed for quantification of naringin and its two metabolites, naringenin and naringenin glucuronide. Naringin and naringenin were extracted from rat plasma with ethyl acetate, using hesperidin as an internal standard. Components in the extract were separated on a 100 mm × 2.0 mm Betabasic 5 μm C₁₈ ODS column by isocratic elution with 70% methanol. The components were analyzed in the multiple-reaction-monitoring (MRM) mode in the precursor/product ion pair of m/z 581.3/273.4 for naringin, m/z 273.4/153.1 for naringenin and m/z 611.5/303.4 for hesperidin, respectively. Linear calibration curves were obtained in the range of 5–1000 ng/ml, using 0.1 ml rat plasma. The within-day coefficients of variation (CVs) were 3.1, 1.8 and 2.2% for naringin, 3.0, 3.3, 3.1% for naringenin at 5, 50 and 500 ng/ml (n = 5). The between-day CVs were 3.4, 1.7 and 4.9% for naringin and 4.0, 3.0, 4.6% for naringenin (n = 5) at 5, 50 and 500 ng/ml respectively. A formulation based on PEG400 was used and orally administered to Sprague-Dawley male rats. Plasma drug concentrations were measured by this method and the pharmacokinetics was analyzed by WinNonlin computer software. Plasma concentration–time profiles of naringin were found to increase quickly and decline rapidly within 2 h and could not be detected after 24 h. Naringenin and naringenin glucuronide occurred slower and the T_max were about 9 and 7.5 h later, respectively.

Introduction

Naringin (4′,5′,7-trihydroxy flavanone-7-rhamnonglucoside) is a flavonoid present in many Citrus fruits and traditional Chinese medicines [1], [2]. Like most flavonoids, naringin has anti-inflammatory [3], anti-ulcer [4], superoxide, and antioxidation [5] activities. Early research has shown that orally administered naringin can be metabolized into naringenin and naringenin glucuronide [6], [7], [8]. Naringenin, the aglycone of naringin, has also been found to exhibit anti-ulcer [4], antioxidant [5], and inhibition of breast cancer proliferation [9]. Several liquid chromatographic methods with UV-detection for detecting naringin in rabbit plasma [6], human plasma [7], human urine [8], and grapefruit juice have been developed. Ishii et al., reported the limits of detection by an assay in human urine was approximately 5 ng for naringin and 1 ng for naringenin [8], which is not sensitive enough to assay the plasma concentration of naringin due to the low oral dosage. To this day, research using the HPLC method with UV-detection to assay the naringin metabolite are unable to assay the naringin and its metabolite naringenin concurrently. Lack of specificity and low sensitivity still limit the effective detection of naringin and its metabolites.

The LC/MS/MS is a method that provides superior selectivity, sensitivity and a rapid rate of analysis. The method is accurate and highly sensitive for detecting plasma concentration in pharmacokinetics which accounts for its increasing popularity [10], [11], [12], [13], [14], [15], [16]. Our research established this new method of using LC/MS/MS for the determination of naringin and its metabolite naringenin concurrently in the plasma of Sprague-Dawley male rats.

Section snippets

Materials and reagents

Naringin and naringenin were purchased from Sigma (St. Louis, MO, purity >95%), while the internal standard (IS) hesperidin, was purchased from the National Institute for the control of pharmaceutical and biological products (HPLC grade). Stock solutions of naringin, naringenin and hesperidin were prepared by dissolving them in methanol. All other chemicals and solvents were of analytical-reagent grade and were used without further purification.

Instrumentation

Analyses were performed using a SHIMADZU HPLC

LC/MS/MS assay

Under ESI conditions, naringin gave MH⁺ at m/z 581.3 as the base ion, naringenin and hesperidin gave MH⁺ at m/z 273.4 and m/z 611.5 as the base ion. The ions m/z 581.3, 273.4 and 611.5 were selected for collision-induced dissociation (CID) experiments. Naringin generated one major product ion at m/z 273.4 (Fig. 1A), naringenin generated two product ions at m/z 147.1 and m/z 153.1 (Fig. 1B), and hesperidin generated two product ions at m/z 449.1 and 303.4 (Fig. 1C).

The precursor/product ion

Discussion

Naringin considered the most potent okadaic acid-antagonistic flavonoid, has been shown to exhibit bioactivity in a number of different ways such as anti-inflammatory [3], anti-ulcer [4], superoxide, and antioxidation [5]. Previous methods were used HPLC-UV to detect the naringin and its metabolites. Compared with the HPLC method, the lower limits of this method for naringin was 0.5 ng/ml, approximately 100 times higher than that of HPLC. Compared with LC/MS/MS, the HPLC method cannot detect

Conclusion

A highly sensitive and specific LC/MS/MS method for the quantification of naringin and its two metabolites was developed and successfully applied to investigate the pharmacokinetics of naringin following oral administration in Sprague-Dawley male rats.

Acknowledgements

Authors are grateful to the SINOVA Medicine Technology Development Limited Company for technical and mass spectrometer facility support.

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Citation Excerpt :
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PM_2.5 is closely related to the incidence and mortality of respiratory diseases. Diesel particulate matter (DPM) is the main component of particulate air pollution and an important source of PM_2.5.
This study mainly explored the effect of DPM on airway surface liquid (ASL) secretion and the regulation of naringin in this process, to evaluate therapeutic potentials of naringin for the treatment of abnormal secretion of the respiratory tract caused by PM_2.5.
The concentration of lysozyme was measured by Lysozyme Assay Kit. Total protein content was determined by the BCA Protein Assay Kit. The concentration of cAMP and MUC5AC, expressions of CFTR, AQP1, and AQP5 proteins were measured by ELISA. Expressions of CFTR, AQP1 and AQP5 mRNA were determined by qPCR. Amount of CFTR on the cell membrane was determined by immunofluorescence.
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These results demonstrated that naringin had regulating effects on the DPM-induced abnormal secretion of the respiratory tract.

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Short communicationA rapid LC/MS/MS quantitation assay for naringin and its two metabolites in rats plasma

Abstract

Introduction

Section snippets

Materials and reagents

Instrumentation

LC/MS/MS assay

Discussion

Conclusion

Acknowledgements

J. Chromatogr.

Life Sci.

J. Chromatogr. B

J. Chromatogr. B

J. Pharm. Biomed. Anal.

J. Pharm. Biomed. Anal.

J. Pharm. Biomed. Anal.

J. Chromatogr. B

Short communication
A rapid LC/MS/MS quantitation assay for naringin and its two metabolites in rats plasma