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050-8705 (1136-1140).pmdDetermination of Atenolol, Rosuvastatin, Spironolactone,
Glibenclamide and Naproxen in Blood by Rapid
Analysis Liquid Chromatography
IN-HAI MA, YONG-FANG PENG, YI-RAN XU , GUANG-YU YANG and QIU-FEN HU Department of Chemistry, Kunming Teacher's College, Kunming 650031, P.R. China. A rapid liquid chromatographic (HPLC) method for the determination of atenolol, rosuvastatin, spirnolactone, glibenclamide and naproxenwas developed. The atenolol, rosuvastatin, spirnolactone, glibenclamideand naproxen were extracted from the sample with acetonitrile and theywere separated on a ZORBAX Stable Bound (4.6 mm × 50 mm, 1.8 µm)C18 rapid analysis column with methanol and 0.01 mol L-1 of acetic acid(78:22) as the mobile phase. This method provides good reproducibilityand sensitivity. The relative standard derivation of overall intra-day variationswere less than 2.5 % and the relative standard derivation of inter-dayvariations were less than 3.0 %. The standard recoveries were rangedfrom 94-103 %.
Key Words: Atenolol, Rosuvastatin, Glibenclamide, Naproxene,
Spironolactone, Rapid analysis liquid chromatography.
The atenolol, rosuvastatin, spirnolactone, glibenclamide and naproxen are the important routine use drug. Atenolol used in the treatment of hypertension, canimprove cardiac function, reduce the symptoms of heart failure1,2. Rosuvastatin is amember of the cholesterol-lowering drug commonly referred to as statins, used forthe treatment of dyslipidemia and other conditions3,4. Naproxen has antiinflammatory,fever-reducing and pain-relieving properties. It is recommended that this medicationbe taken with food to minimize stomach irritation5,6. Glibenclamide is widely usedfor the treatment of non-insulin-dependant diabetes mellitus (NIDDM) diabetestype. This agent has been reported to inhibit the activities of various ion channelsand transporters7,8. Spironolactone is a potassium-sparing diuretic and is marketedas a competitive aldosterone antagonist. It is indicated for the treatment of oedemaand ascites in cirrhosis of the liver and has been widely utilized in the managementof congestive heart failure associated with congenital heart disease, bronchopul-monary dysplasia or chronic lung disease and pediatricascites9,10.
†De partment of Chemistry, Yunnan Nationalities University, Kunming 650031, P.R. China Determination of Drugs in Blood by Rapid Analysis LC 1137 Several works on the HPLC methods for the quantitation of these drugs in human plasma was reported11-17. However, these methods usually need a tedioussamples preparation or long time for chromatographic separation. In this paper, asimple, rapid and sensitive HPLC analytical method using ZORBAX stable boundrapid analysis column for the simultaneous determination of the atenolol,rosuvastatin, spirnolactone, glibenclamide and naproxen in blood was developed.
Five drugs can achieve baseline separation with 2.0 min. Compared to the routineliquid chromatography method11-17, 80 % of separation time was saved. It is one ofthe most rapid methods for chromatographic simultaneous analysis of atenolol,rosuvastatin, spirnolactone, glibenclamide and naproxen.
The HPLC analysis was performed on a Waters 2695 Alliance separation system equipped with a Waters 996 photodiode array detector (Waters Corporation, Milford.,MA 01757, USA). A ZORBAX stable bound column (4.6 mm × 50 mm, 1.8 µm)(Agilent Technologies Inc, Santa Clara., CA 95051, USA) was utilized.
The atenolol, rosuvastatin, spirnolactone, glibenclamide and naproxen reference standards were obtained from Shanghai Usea Biotech Company (Shanghai, P.R.
China). They purities were ≥ 98 %. HPLC grade acetonitrile and methanol wereprovided by Fisher Scientific Inc (Madison, WI 53711, USA). The ultrapure waterused was obtained from a Milli-Q50 SP Water system (Millipore Inc, MA 01730Bedford). The mobile phase used is methanol and 0.01 mol L-1 of acetic acid (78:22)at a flow-rate of 2.0 mL min-1. The sample injection volume is 20 µL. The detectionwavelength was 235 nm. The chromatogram of drug standards and blood sample at235 nm is shown in Fig. 1.
Preparation of sample: Multiple blood samples (10 mL) of 10 healthy non-
smoker volunteers (ages ranging from 22-25 years) not involved in any strenuousactivity and not taking any other medications were collected in evacuated glasstubes. The blood was then centrifuged at 5000 rpm for 10 min, the plasma separatedand deproteinated by acetonitrile. The supernatant obtained was filtered through a0.45 µm filter. Serum thus obtained was stored at -20 ºC pending drugs analysis.
Preparation of standard solution: To prepare standard solutions, an accurately
weighed amount of atenolol, rosuvastatin, spirnolactone, glibenclamide andnaproxen which were dissolved in methanol for HPLC. Five concentrations werechosen, with the range 0.05-150 µg mL-1, respectively. The regression equations ofthe five drugs were established based on the standard samples injected and theirpeaks area.
RESULTS AND DISCUSSION
To optimize the HPLC conditions for the separation of atenolol, rosuvastatin, spironolactone, glibenclamide and naproxen, the effects of pH, mobile phase compo-sition, the type of column and its dimension and wavelength of detection wereinvestigated.
The first factor examined was the type of the stationary phase. C8 and C18 columns of different dimensions and particle sizes were used. It was found ZORBAX stablebound C18 rapid analysis column (4.6 mm × 50 mm, 1.8 µm) gave the most suitableresolution. By this rapid analysis column, the five drugs were separated completelywithin 2.0 min (Fig. 1). Compared to the previous literature11-17, this is one of themost rapid methods to simultaneous separation of the five drugs.
Fig. 1. HPLC chromatogram of drug standards (b) and blood sample (a) The mobile phase compositions were prepared with appropriate ratios of methanol or water. The adjustment of pH was made using drops of acetic acid, to achieve pH3.0-4.5. It is observed that the best resolution of all drugs was achieved with amobile phase composed of methanol and 0.01 mol L-1 of acetic acid (78:22, v/v).
The use of methanol and water showed an elevated base line and no tailing effect isobserved.
The absorption spectrum of atenolol, rosuvastatin, spirnolactone, glibenclamide and naproxen were obtained with 996 photodiode array detector. Results show thatthe analyts have the strong absorption at 235 nm. Therefore, the 235 nm was selectedas detecting wavelength.
Plasma deproteination for the determination of drugs is commonly accepted as the simplest method of sample preparation. Previously developed HPLC proceduresfor the determination of atenolol, rosuvastatin, spironolactone, gilibenclamide andnaproxen in plasma are based on liquid-liquid extraction or solid-phase extractionfrom plasma samples. The method applied in present study involved the direct injectionof the plasma samples after precipitation of protein with acetonitrile. This does notneed a complex purification procedure. The sample preparation was greatly simplified.
Determination of Drugs in Blood by Rapid Analysis LC 1139 Under the optimum conditions, the regression equations of five drugs were established based on the standard samples injected and their peaks area. The limitsof detection are calculated by the ratio of signal to noise (S/N=3). The results wereshown in Table-1. The reproducibility of this method was also examined for 1.0 mg mL-1of the five drugs. The relative standard deviations (n = 9) were shown in Table-1.
REGRESSION EQUATION, COEFFICIENT AND DETECTION LIMIT The recovery test were carried out by adding atenolol, rosuvastatin, spirono- lactone, gilibenclamide and naproxen to the samples (three different concentrationsof markers: 0.5, 1.0 and 5.0 µg). The sample was prepared and injected for HPLCanalysis to calculate the amount of the drugs founded. The results shown that therecoveries (n = 5) were ranged from 94-103 %. This method is high recovery.
The measurements of intra- and inter-day variability (determination of the same samples for seven times) were utilized to evaluate the precision of the developedmethod. The results shown that the relative standard derivation of overall intra-dayvariations were less than 2.5 % and the relative standard derivation of inter-dayvariations were less than 3.0 %.
The proposed HPLC method enables simultaneous determination of atenolol, rosuvastatin, spironolactone, glibenclamide and naproxen because of good separationand resolution of the chromatographic peaks within 2 min. Compared to the normalcolumn, more than 80 % of separation time was saved. It is one of the most rapidmethods for chromatographic analysis of the five drugs. The sample can directlyafford to HPLC analysis after precipitation of protein with acetonitrile. This prepa-ration does not need a complex purification procedure. The sample preparation wasgreatly simplified. The detector response was found to be linear over a wide concen-tration range for all drugs. The validity, LOQ and the linearity range of the methodmakes it acceptable method for clinical studies in patients taking these medicationssimultaneously. Results are accurate and precise and are confirmed by the statisticalparameters and are well within the limits required for bioanalytical assays. Thelower limit of quantification permits the use of the method for pharmacokineticstudies.
Thus, this method is rapid, high sensitive and selective and provides good repro- ducibility and accurateness for the quantification of these five drugs.
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(Received: 14 January 2009; Accepted: 16 October 2009) AJC-7960
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