View More View Less
  • 1 University of Petra, Amman, Jordan
  • 2 The Jordanian Pharmaceutical Manufacturing Company (PLC), Naor 11710, Jordan
Open access

Krebs buffer is considered one of the most used physiological buffers in biomedical research. In the current work, a rapid reversed-phase high-performance liquid chromatographic (RP-HPLC) method with ultraviolet (UV) detection at 214 nm was developed and validated according to European Medicines Evaluation Agency (EMEA) guidelines for the determination and quantification of propranolol in Sprague–Dawley rat's serum and in Krebs buffer. This method can be applied for both in vivo and in vitro studies with short run time of 7.0 min . Isocratic elution with a flow rate of 1.0 mL/min was employed. BDS Hypersil C-18 column (150 mm × 4.6 mm and 5 μm) was used to obtain satisfactory resolution. The mobile phase used contained a mixture of acetonitrile, methanol, and triethylammonium phosphate solution (15.0:32.5:52.5, v/v). Best separation between propranolol and the internal standard (I. S.) sildenafil was obtained at 4.2 and 5.5 min, respectively. Propranolol was linear over a concentration range of 50.00–3000 ng/mL with acceptable accuracy, and intra- and inter-day precision. Dilution integrity was assessed and was found to be within the acceptable range for both serum and Krebs buffer. Sample stability tests were studied at different storage conditions, and all the analytes were found to be stable. The mean percentage of recovery of propranolol was found to be 97.06% and 98.57% for serum and Krebs buffer, respectively.

Abstract

Krebs buffer is considered one of the most used physiological buffers in biomedical research. In the current work, a rapid reversed-phase high-performance liquid chromatographic (RP-HPLC) method with ultraviolet (UV) detection at 214 nm was developed and validated according to European Medicines Evaluation Agency (EMEA) guidelines for the determination and quantification of propranolol in Sprague–Dawley rat's serum and in Krebs buffer. This method can be applied for both in vivo and in vitro studies with short run time of 7.0 min . Isocratic elution with a flow rate of 1.0 mL/min was employed. BDS Hypersil C-18 column (150 mm × 4.6 mm and 5 μm) was used to obtain satisfactory resolution. The mobile phase used contained a mixture of acetonitrile, methanol, and triethylammonium phosphate solution (15.0:32.5:52.5, v/v). Best separation between propranolol and the internal standard (I. S.) sildenafil was obtained at 4.2 and 5.5 min, respectively. Propranolol was linear over a concentration range of 50.00–3000 ng/mL with acceptable accuracy, and intra- and inter-day precision. Dilution integrity was assessed and was found to be within the acceptable range for both serum and Krebs buffer. Sample stability tests were studied at different storage conditions, and all the analytes were found to be stable. The mean percentage of recovery of propranolol was found to be 97.06% and 98.57% for serum and Krebs buffer, respectively.

Introduction

Propranolol is chemically described as 2-propanol, 1-[(1-methylethyl) amino]-3-(1-naphthalenyloxy) (Figure 1). Propranolol is a highly lipophilic drug that is almost completely absorbed from the gastrointestinal tract following oral administration. It is present as a white crystalline powder with a pKa value of 9.4 [1]. Propranolol is the prototype of all non-selective β-adrenergic blockers with no intrinsic sympathetic activity. It is used for the treatment of many cardiovascular diseases such as hypertension, arrhythmias [2, 3], and angina pectoris [4]. Recent studies have shown that propranolol is the first-line treatment for infantile hemangiomas [57] and is used in infants to treat congestive heart failure with congenital heart disease [5]. Moreover, propranolol is still widely used by clinicians because of its high efficacy and low cost [3]. Accordingly, it is listed by World Health Organization's List of Essential Medicines as one of the drugs used for a basic health system [6].

Figure 1.
Figure 1.

Chemical structure of propranolol and sildenafil

Citation: Acta Chromatographica Acta Chromatographica 30, 3; 10.1556/1326.2017.00018

Different high-performance liquid chromatography (HPLC) methods were developed for the determination and quantification of propranolol in various biological fluids such as blood, serum, plasma, and urine. Consequently, many HPLC–ultraviolet (UV) methods as well as HPLC–fluorescence or mass spectroscopic methods were developed for the determination of propranolol in human's and rat's serum [79], in the plasma [1, 914], in rabbit's plasma [15], and in dog's plasma [8, 16]. However, many of those methods require a volume of plasma more than 100 μL [11, 16, 17] as well as multiple steps of sample preparation and extraction procedures being a time-consuming methods [1, 7, 16, 17]. Moreover, none of the previous methods measured propranolol in Krebs buffer, a physiological solution, which has been used as a bathing and perfusion medium providing the cell with the essential ions that maintain the pH of the cell within the physiologic range for in vitro studies [18, 19].

The aim of this work is to develop a rapid, accurate, precise, reliable, and a simple one-step protein precipitation HPLC–UV method validated according to European Medicines Evaluation Agency (EMEA) guidelines in order to obtain a feasible analytical method. This method can be applied successfully for both in vivo and in vitro pharmacokinetic and pharmacodynamics studies of small concentrations of propranolol (50.00 ng/mL) in Sprague Dawley rat's serum and in Krebs buffer experiments.

Experimental Part

Reagents and standards

All solvents used were HPLC grade, and all chemicals were reagent grade. Methanol, acetonitrile, monobasic potassium phoshate, and deionized water (Nanopure™) were all obtained from Fisher Scientific (Loughborough, Leicestershire, UK), phosphoric acid was obtained from Kyowa Medex Co. (Tokyo, Japan), and trimethylamine was purchased from TEDIA Company (Tedia Fairfield, OH, USA). Propranolol hydrochloride was a kind gift from The Arab Pharmaceutical Manufacturing (APM, Salt, Jordan), while Sildenafil citrate, the internal standard (I. S.), was kindly obtained from Jordan Pharmaceuticals Manufacturing (JPM, Amman, Jordan). Potassium chloride and ethylene diamine tetra acetic acid were purchased from Acros Organics (Acros Organics BVBA, Geel, Belgium). Sodium chloride, anhydrous calcium chloride, magnesium sulfate, potassium dihydrophosphate, sodium bicarbonate, and d-(+)-glucose were all purchased from (Sigma-Aldrich, St. Louis, Missouri).

Instrumentation

The study was carried out on HPLC system Finnigan Surveyor (Thermo Electron Corporation, San Jose, CA, USA) comprised of the pump (solvent delivery systems pump) (LC Pump), autosampler (Autosampler Plus), coupled with BDS hypersil™ C-18 Column (150 mm × 4.6 mm, 5 μm) (Thermo Electron Corporation, San Jose, CA, USA), and the detector (UV-VIS Plus Detector). Computer system used was Windows XP, and the software used was ChromQuest software 4.2.34.

Analytical and chromatographic conditions

Chromatographic analysis was carried out at ambient temperature 22–25 °C which resembles sample processing temperature during analysis stages, whereas column and autosampler temperature were maintained at 40.0 and 4.0 °C, respectively. Compounds were separated isocratically at a flow rate of 1.0 mL/min. Samples were injected using a volume of 15 μL. Short run time of 7.0 min was applied. The effluent was monitored spectrophotometrically at wavelength 214 nm.

Preparation of the mobile phase

Mobile phase used consisting of a mixture of acetonitrile, methanol, and triethylammonium phosphate solution (15.0:32.5:52.5, v/v) as well as triethylammonium phosphate solution was prepared by adding 900 μL triethylamine to 1.0 L of water, and then, the pH was adjusted to 2.75 using phosphoric acid.

Preparation of stock solutions

Stock solutions of propranolol and the I. S. sildenafil were prepared separately to obtain a final concentration of 200.0 μg/mL and 10,000 μg/mL, respectively.

Preparation of working solutions

Preparation of the working solution of the internal standard

Sildenafil stock solution (250 μL) was diluted to 50 mL of acetonitrile to obtain 5.0 μg/mL of sildenafil (I. S.) working solution.

Preparation of working spiking solutions for calibration and quality control samples

Calibration curves were constructed to obtain the following concentrations: 50.0, 100.0, 200.0, 500.0, 1000, 2000, and 3000 ng/mL, whereas quality control (QC) samples concentrations were 50.00, 150.0, 1500, and 2500 ng/mL for lower limit of quantification (LLOQ), QC low, QC mid, and QC high, respectively. Working solutions of the calibration curve and QC samples were prepared by taking different volumes from propranolol stock solution of 10.0, 20.0, 40.0, 100.0, 200.0, 400.0, and 600.0 μL, respectively, for calibration curve preparation and 10.0, 30.0, 300.0, and 500.0 μL, respectively, for QC samples preparation. The volume was completed up to 1.0 mL final volume. Finally, serum and Krebs buffer spiking samples were prepared by taking 25.0 μL of each working solution to be spiked resulting in 1.0 mL final volume.

Method of extraction

An appropriate number of Eppendorf tubes were placed in a rack and labeled properly. Next, 100 μL aliquots of each serum or Krebs buffer test samples, spiked calibration curve samples, and QC samples were pipetted into the appropriate labeled tube. Later, 150 μL of the I. S. was added. The I. S. sildenafil was dissolved in the organic solvent acetonitrile to precipitate proteins for the one-step extraction. Finally, each sample was vortexed vigorously for 1.0 min, centrifuged at 18.407 × g for 15 min and injected into the HPLC system to be analyzed.

Preparation of Krebs buffer

Krebs bufferwas prepared by mixing the following volumes (in mL) of 1.0 M: NaCl 118, KCl 4.5, KH2PO4 1.2, NaHCO3 25.0, MgSo4 1.60, glucose 5.5, CaCl2 2.5, and 0.25 mL of 0.1 M EDTA; the volume was completed to 1.0 L by distilled water, and then, the buffer was oxygenated with oxygen concentrator (dual flow oxygen concentrator for oxygen bar, Hebei, China). CaCl2 was added after oxygenation to prevent turbidity; pH was then adjusted to 7.4 using 1.0 M HCl (0.08 mg/mL).

Validation of the method

Validation was conducted according to EMEA guidelines of bioanalytical method validation [20].

To confirm the suitability of the method, it was validated for specificity, accuracy, intra- and inter-day precision, stability, linearity, dilution integrity, recovery, limit of detection, and lower limit of quantification.

Specificity

The specificity of the method was determined by analyzing six rat blank serum and blank Krebs buffer samples to prove the absence of interference on the analysis.

Accuracy and precision

Accuracy, and intra- and inter-day precision were evaluated at four levels of concentrations by analyzing six replicates (n = 6) of each QC concentration: LLOQ, QC low, QC mid, and QC high on three different days accompanied by a calibration curve for each day in order to calculate the concentration per each. Accuracy was expressed as % recovery (measured value/theoretical value × 100), whereas intra-day and inter-day precisions were expressed as the relative standard deviation (RSD) (square root of the residual variance/the mean × 100). According to EMEA guideline, % recovery of QC (low, mid, and high) should be within 85–115% while LLOQ should be within 80–120%; in regard to RSD value, it should not exceed 15% for all QC samples (QC low, mid, and high), except for the LLOQ which should not exceed 20% [20]. All results obtained between the 3 days were similar and acceptable.

Stability

Sample storage stability

Stability was conducted to ensure that every step taken during sample preparation, storage, and analysis will not affect the concentration of the analyte. QC low and QC high were used to evaluate analyte stability in serum and in Krebs buffer immediately after preparation and after applying storage conditions to the samples to be evaluated. Storage conditions applied were stock solution stability, freeze and thaw stability, bench-top stability, autosampler stability, and long-term stability; % recovery of QC low and QC high should be within 85–115% according to EMEA guidelines [20].

Freeze and thaw stability

QC low and QC high samples were spiked properly in serum and in Krebs buffer without carrying out the extraction procedure.

With corresponding calibration curve at zero time, QC samples were extracted and analyzed, and the resultant concentrations were calculated. The remaining spiked samples were kept frozen at −20 °C. After the first and the second cycle, samples were thawed at room temperature (25 °C) and then were refrozen again. Finally, after the last (third) cycle, spiked samples were thawed and then extracted to be analyzed and the resultant concentrations were calculated.

Bench-top stability

QC low and QC high samples were spiked properly in serum and in Krebs buffer without carrying out the extraction procedure.

With corresponding calibration curve at zero time, QC samples were extracted to be analyzed and the resultant concentrations were calculated. The remaining spiked samples were left on the bench at room temperature 25 °C. After 24 h, samples were extracted and analyzed and the resultant concentrations were calculated.

Autosampler stability

At zero time, calibration curve was freshly prepared; QC low and QC high samples were also prepared using sufficient volume for the test. All the samples were spiked properly in serum and in Krebs buffer, extracted, and then analyzed, and the resultant concentrations were calculated. QC samples were left in the autosampler for 24 h and then were analyzed again, and the resultant concentrations were calculated to determine % recovery.

Long-term stability

Initially, QC low and QC high samples were spiked properly in serum and in Krebs buffer without carrying out the extraction procedure. Next, with corresponding calibration curve at zero time, QC samples were extracted and analyzed, and the resultant concentrations were calculated. The remaining spiked samples were stored in the freezer at −20 °C under the same storage conditions for study samples for 30 days.

Stock solution stability

Six aliquots of propranolol stock solution were analyzed at zero time and after applying storage conditions for 24 h at room temperature as well as after storage in refrigerator at 4 °C for 1 week and 1 month. Similarly, six aliquots of I. S. stock solution were analyzed after storage for 24 h at room temperature, and after storage for 1 week and 1 month at 4 °C. After each storage period, propranolol and sildenafil concentrations were related to the initial concentration that was determined for freshly prepared samples [20].

Linearity

Each calibration curve contained seven standard concentrations (50.00, 100.0, 200.0, 500.0, 1000, 2000, and 3000 ng/mL). Six calibration curves were performed throughout method validation. Depending on the data obtained from those six calibrations; linearity curve was obtained by plotting the mean ratios of the six calibration standards versus each concentration level to obtain the correlation coefficient (R2). Correlation coefficient value should be more than 0.98. Least-squares linear regression analysis of the calibration data was performed using the linear equation y = mx + c [20].

Dilution integrity

Accuracy and precision should not be affected by the dilution of samples. The matrix was spiked with an analyte concentration above the upper limit of quantification (ULOQ) and then was diluted with blank matrix (six determinations per dilution factor). Accuracy and precision should be within the acceptable range according to EMEA guidelines. Dilution integrity should cover the dilution applied to the study samples [20].

Absolute recovery

Drug and I. S. recovery

Two groups of QC low, mid, and high were prepared. In the first group, both propranolol and the I. S. were prepared in the mobile phase which are unextracted samples with 100% recovery, whereas the second group was spiked and extracted in serum and in Krebs buffer (n = 3). RSD values were calculated from the resultant area under the curve (AUC) of both the drug and the I. S. followed by the calculation of the absolute recovery for each of them [20].

The limit of detection and quantification

The lower limit of detection (LLOD) and the lower limit of quantification (LLOQ) were estimated by the baseline noise method. LLOD was estimated at a signal-to-noise ratio (S/N) of 3, whereas LLOQ was estimated at a signal-to-noise ratio (S/N) of 10. The calculated LLOD and LLOQ were 15 and 50 ng/mL, respectively.

Results and discussion

Method development

Propranolol is a highly lipophilic drug that is classified by the Biopharmaceutics Classification System (BCS) as being a drug with high solubility and high permeability [1, 21]. A series of experiments was tested in our laboratory to develop a sensitive, accurate, one-step extraction method for quantitative analysis of propranolol. By referring to previous HPLC methods developed, propranolol was detected in plasma using reversed-phase HPLC method. Furthermore, because of propranolol lipophilicity, UV detectors and, hence, HPLC–UV methods were selected in many of the studies to detect propranolol in plasma and in serum [1, 7, 14, 15, 22]. UV detectors are selected not only because of being inexpensive but also they tend to be the first choice for lipid analysis because they are able to support continuously variable wavelengths. Thus, they can be considered a great advantage for lipid analysts during method development [23]. However, many RP-HPLC–UV methods require high volume of blood which is difficult to be obtained from rat's blood. For example, 5 mL of human plasma [24], 2 mL of human plasma [7], 1 mL of rabbit plasma and human plasma [1, 15], and 500 μL of dog plasma [16] were collected. Accordingly, none of these methods used 100 μL of blood which is the reasonable amount that can be collected from rats. Moreover, none of those methods were with a run time less than 7 min. Nevertheless, many of these studies have low LLOQ with a concentration range between 20–280 [24], 40–2000 [7], 2–500 [16], and 15–180 ng/mL [1], except for Saharty and his colleagues with a higher concentration range of 5–200 μg/mL which is not useful for clinical pharmacokinetic studies. However, multiple steps of extraction procedures were required to obtain satisfactory results in many of these methods [1, 7, 16]; thus, it may affect the recovery and stability of the method and may be a time-consuming method. Another RP-HPLC method with a diode array detector used 1 mL of human urine for the simultaneous determination of propranolol along with other 22 drugs and obtained an LLOQ and LLOD of 450 and 130 ng/mL, respectively. As for fluorescence and mass spectrometry detectors, also high volume of biological fluids was used; for instance, 200 μL of human serum/plasma [9] and 0.5 mL of human plasma [11, 22] were used. Because fluorescence and mass spectrometry achieve a high level of sensitivity and specificity as well as high level of qualitative capacity, the LLOQ was obtained by these detectors with a concentration range between 5–200 [9], 1–100 [22], 0.3–300 [11], 2–800 [17], 3.13–100 [10], and 10–100 ng/mL [13].

Selection of the internal standard

Selection of the internal standard depends on chemical similarity to propranolol, UV absorption at 214 nm, stability, comparable retention times, and similarity in the extraction procedures for both of the drugs [25]. Good separation between propranolol and sildenafil was obtained at 4.2 and 5.5 min, respectively.

Selection of the column

Different C8 and C18 columns with different lengths, temperatures, and particle sizes were tested. Initially, a BDS hypersil C8 column (100 mm × 4.6 mm, 3 μm) was tested, yet a clear fronting peak was overlapped with propranolol in serum but not in Krebs buffer. To avoid such interference, the polarity of the column was reduced and the C18 column was tested to obtain a better separation. Consequently, a BDS hypersil C18 (100 mm × 4.6 mm, 5 μm) was examined, inappropriately; a small interfering peak overlapped with the small concentrations of propranolol, mainly LLOQ. Accordingly, the reproducibility and sensitivity of LLOQ were affected. Thus, to increase the efficiency of the separation, column length was increased. Longer columns (150–300 mm) are a good choice because it always offers a greater resolution and avoids fronting peaks due to the increased analysis time. Finally, the best peak shape, resolution, efficiency, and reproducibility were obtained by using BDS hypersil C18 column (150 mm × 4.6 mm, 5 μm). However, the separation between propranolol and the internal standard (sildenafil) was dissatisfactory at room temperature (25 °C). Therefore, the temperature was optimized at 40 °C instead of 25 °C, yielding better solubility and diffusivity with an agreeable symmetrical peak shape and widths.

Sample preparation

An easy, one-step protein denaturation was selected in our method since it is more efficient and more rapid. Acetonitrile was used in our method because it is a common solvent for HPLC as it precipitates 95% of proteins [26]. Thus, as the polarity of an organic solvent decreases, it becomes more effective and easier to precipitate proteins. Therefore, acetonitrile was used because it has the advantage of being less polar than other organic solvents such as methanol [27]. Accordingly, protein precipitation method with acetonitrile was found to be optimal in our method for the extraction of propranolol in serum and in Krebs buffer.

Selection of appropriate wavelength

Several preliminary experiments were carried out to achieve the best peak shape with the shortest retention time. Many wavelengths were tested, 250, 290, and 214 nm, resulting in the best detection at 214 nm wavelength with adequate sensitivity.

Assay validation

Specificity

No peaks were observed at the retention times of propranolol and sildenafil; thus, specificity was confirmed (Figure 2).

Figure 2.
Figure 2.

Representative chromatograms of blank and spiked propranolol samples (LLOQ) (50 ng/mL) in (A) serum and (B) Krebs buffer and extracted with sildenafil (5 μg/mL); the internal standard of propranolol analysis

Citation: Acta Chromatographica Acta Chromatographica 30, 3; 10.1556/1326.2017.00018

Accuracy

Overall range of % recovery for the concentrations for days 1, 2, and 3 was within (99.5–107.5) for serum and (103.2–107.4) for Krebs buffer. On the other hand, overall % recovery results obtained for the three days (inter-day) were within (101.1–104.7) for serum and (103.9–106.6) for Krebs buffer (Table 1).

Table 1.

Accuracy, and intra- and inter-day precision for the determination of propranolol in rat serum and Krebs buffer

Concentration (ng/mL)Rat serum
Intra-day (n = 6)Inter-day (n = 18)
Day 1Day 2Day 3Day 1Day 2Day 3Day 1Day 2Day 3Mean value of measured concentrations ± SDRecovery (%)RSD (%)
Mean value of measured concentration ± SDRecovery (%)RSD (%)
LLOQ (50.0)52.1 ± 1.253.7 ± 1.151.2 ± 0.5104.2107.5102.32.32.01.052.3 ± 1.3104.72.7
QC low (150.0)149.3 ± 1.9151.9 ± 2.9153.6 ± 3.399.5101.3102.41.31.92.1151.6 ± 2.2101.12.1
QC mid (1500)1543.9 ± 11.01504.8 ± 14.11504.2 ± 27.2102.9100.3100.30.70.91.81517.6 ± 22.7101.21.7
QC high (2500)2610.6 ± 22.52538.1 ± 16.92540.7 ± 71.3104.4101.5101.60.90.72.82563.2 ± 41.1102.52.1
Concentration (ng/mL)Krebs buffer
Intra-day (n = 6)Inter-day (n = 18)
Day 1Day 2Day 3Day 1Day 2Day 3Day 1Day 2Day 3Mean value of measured concentrations ± SDRecovery (%)RSD (%)
Mean value of measured concentration ± SDRecovery (%)RSD (%)
LLOQ (50.0)51.7 ± 2.152.8 ± 1.252.6 ± 2.3103.4105.7105.24.12.34.452.4 ± 0.6104.84.0
QC low (150.0)156.6 ± 7.1156.4 ± 6.3156.5 ± 7.0104.4104.3104.34.54.04.5156.5 ± 0.1104.44.1
QC mid (1500)1610.4 ± 20.91581.1 ± 55.81603.5 ± 22.4107.4105.4106.91.33.51.41598.3 ± 15.3106.62.3
QC high (2500)2596.6 ± 37.32612.6 ± 63.42580.3 ± 68.4103.9104.5103.21.42.42.72596.5 ± 16.1103.92.2

Precision

The RSD values measured for all tested concentrations of intra-day validation were found to range between (0.7–2.8) for serum and (1.3–4.5) for Krebs buffer. On the other hand, RSD values obtained from the repeatability study of inter-day validation ranged from 1.7 to 2.7 for serum and 2.2 to 4.1 for Krebs buffer. These results showed that this method is repeatable (Table 1).

Sample storage stability

Propranolol stability in solution and after sample preparation in serum and in Krebs buffer was evaluated. Spiked samples of QC low and QC high were prepared in triplicate. No change in propranolol % recovery or in the chromatographic behavior was observed. Similarly, no change in the chromatographic behavior of propranolol or sildenafil was observed. All stability results of freeze–thaw, bench-top, autosampler, and long-term stability were within the accepted range in accordance to EMEA guidelines (Tables 2 and 3) [20].

Table 2.

Sample storage stability mean % recovery results of freeze–thaw stability, bench-top stability, autosampler stability, and long-term stability data in rat's serum and Krebs buffer

Freeze–thaw stabilityBench-top stabilityAutosampler stabilityLong-term stability
Serum (% recovery)Time0.0 h3 Cycles24.00 h24.00 h30 days
QC low (150.0 ng/mL)98.9 ± 1.898.3 ± 2.296.5 ± 1.398.5 ± 1.598.0 ± 1.6
QC high (2500 ng/mL)102.3 ± 1.4103.2 ± 1.4102.1 ± 1.3102.9 ± 0.998.6 ± 2.0
Krebs buffer (% recovery)Time0.00 h3 Cycles24.00 h24.00 h30 days
QC low (150.0 ng/mL)104.4 ± 1.6100.6 ± 1.8103.3 ± 2.9100.9 ± 1.298.4 ± 2.1
QC high (2500 ng/mL)102.6 ± 2.4102.8 ± 1.5103.2 ± 1.2100.7 ± 5.298.2 ± 1.9
Table 3.

Sample storage stability mean % recovery results of stock solution stability

Theoretical concentration (ng/mL)
Propranolol (1000)I. S. (3000)
Recovery (%)RSD (%)Recovery (%)RSD (%)
Time0.00 h (n = 6)100.04.5100.04.0
24.00 h at RT (n = 6)98.12.699.31.3
1 week (4 °C) (n = 6)99.22.998.22.5
1 month (4 °C) (n = 6)98.11.097.91.4

Stock solution stability

Propranolol and sildenafil stock solutions containing 1000 ng/mL and 3000 ng/mL, respectively, were analyzed after storage for 24 h at room temperature and at 4 °C in the refrigerator. All results obtained suggested that propranolol and sildenafil standard solution were stable (Table 3).

Linearity

Linearity was studied over a concentration range of 50.0–3000 ng/mL. Calibration curves of propranolol were analyzed, and correlation coefficients were calculated. R2 values were more than 0.99 for all six calibrations and for linearity curves of propranolol in serum and in Krebs buffer (Table 4).

Table 4.

R 2, slope, and intercept data for the linearity curves of all calibration curves in both serum and Krebs buffer

(n = 6)(R2 )Slope ± SDIntercept ± SD
Serum linearity curve0.99970.0003221 ± 0.000004232−0.006904 ± 0.0007877
Krebs buffer linearity curve0.99910.0002941 ± 0.0002859−0.009996 ± 0.0006550

Dilution integrity

Dilution integrity was assessed by diluting samples prepared at a concentration more than the ULOQ of 5000 ng/mL (2-fold) and 10,000 (4-fold) of n = 6. % Recovery data for propranolol integrity of dilution are presented in Table 5.

Table 5.

Accuracy data for 2× and 4× dilution integrity for propranolol in rat's serum and in Krebs buffer

Theoretical Concentration (ng/mL)Mean value of measured concentration ± SDRecovery (%)RSD (%)
Dilution integrity in serum5000.04925.8 ± 67.498.51.4
10,000.09923.3 ± 158.599.21.6
Dilution integrity in Krebs buffer5000.05115.8 ± 120.4102.32.4
10,000.09910 ± 138.199.11.4

Absolute recovery (extraction coefficient)

Extraction recovery was determined by comparing the peak heights of extracted blood samples and the extracted Krebs buffer samples with the peak heights of standards prepared in the mobile phase. Table 6 shows absolute recovery of propranolol and the I. S. in serum and Krebs buffer with quantitative recovery ranged between 96.0–97.9% and 97.9–99.2% for propranolol in serum and Krebs buffer, respectively. As for I. S., quantitative recovery was 96.2% and 98.4% for serum and Krebs buffer, respectively.

Table 6.

Absolute recovery ratios of propranolol and sildenafil (I. S.) in (A) serum, and (B) Krebs buffer

A
ConcentrationAbsolute recovery (%) ± SD
Absolute recovery of propranolol in the serum
150.0 ng/mL QC low97.2 ± 0.7
1500 ng/mL QC mid96.0 ± 1.2
2500 ng/mL QC high97.9 ± 0.9
Mean absolute recovery of sildenafil (I. S.) in the serum96.2 ± 1.3
B
Absolute recovery of propranolol in Krebs bufferConcentrationAbsolute recovery (%) ± SD
150.0 ng/mL QC low98.7 ± 1.2
1500 ng/mL QC mid99.2 ± 0.8
2500 ng/mL QC high97.9 ± 1.2
Mean absolute recovery of sildenafil (I. S.) in Krebs buffer98.4 ± 0.9

Conclusions

A complete RP-HPLC–UV method was developed for the determination and quantification of propranolol in rat's serum and Krebs buffer. It is a precise, accurate, rapid, stable, linear, and acceptable method according to EMEA guidelines with one-step sample preparation and short run time of 7 min. This method can be applied to pharmacokinetic and pharmacodynamics studies of propranolol using small volume of serum and Krebs buffer (100 μL) for both in vivo and in vitro studies.

Abbreviations

RP-HPLC reversed-phase high-performance liquid chromatographySTD standard deviation RSD relative standard deviation EMEA European Medicines Evaluation Agency I. S. internal standard LLOQ lower limit of quantification ULOQ upper limit of quantification QC quality control R2 correlation coefficient UV ultraviolet

Conflict of interest

The authors declare that they have no conflict of interest.

References

  • 1.

    Salman, S. A.; Sulaiman, S. A.; Ismail, Z.; Gan, S. H. Toxicol. Mech. Methods 2010, 20, 137.

  • 2.

    Cole, S. W.; Sood, A. K. Clin. Cancer Res. 2012, 18, 1201.

  • 3.

    Priviero, F. B.; Teixeira, C. E.; Claudino, M. A.; De Nucci, G.; Zanesco, A.; Antunes, E. Eur. J. Pharmacol. 2007, 571, 189.

  • 4.

    Hebb, A. R.; Godwin, T. F.; Gunn, R. W. Can. Med. Assoc. J. 1968, 98, 246.

  • 5.

    Bruns, L. A.; Canter, C. E. Paediatr Drugs 2002, 4, 771.

  • 6.

    WHO, , World health organization model list of essential medicines: 17th list. Available at http://apps.who.int/iris/bitstream/10665/70640/1/a95053_eng.pdf, 2011. Access date: 1 October 2015.

  • 7.

    Hackett, L.; Dusci, L. Clin. Toxicol. 1979, 15, 63.

  • 8.

    Semple, H. A.; Xia, F. J. Chromatogr. B Biomed. Sci. Appl. 1994, 655, 293.

  • 9.

    Rekhi, G. S.; Jambhekar, S. S.; Souney, P. F.; Williams, D. A. J. Pharm. Biomed. Anal. 1995, 13, 1499.

  • 10.

    Braza, A. J.; Modamio, P.; Mariño, E. L. J. Chromatogr. B Biomed. Sci. Appl. 2000, 738, 225.

  • 11.

    Zhanga, J.; Dinga, L.; Wenb, A.; Wua, F.; Suna, L.; Yangb, L. Asian J. Pharm. Sci. 2009, 4, 169.

  • 12.

    Li, S.; Liu, G.; Jia, J.; Liu, Y.; Pan, C.; Yu, C.; Cai, Y.; Ren, J. J. Chromatogr. B 2007, 847, 174.

  • 13.

    Umezawa, H.; Lee, X. P.; Arima, Y.; Hasegawa, C.; Izawa, H.; Kumazawa, T.; Sato, K. Biomed. Chromatogr. 2008, 22, 702.

  • 14.

    Delamoye, M.; Duverneuil, C.; Paraire, F.; de Mazancourt, P.; Alvarez, J.-C. Forensic Sci. Int. 2004, 141, 23.

  • 15.

    El-Saharty, Y. J. Pharm. Biomed. Anal. 2003, 33, 699.

  • 16.

    Xiao, S.; Wei, G.; Guo, H.; Liu, H.; Liu, C. Asian Journal of Pharmacodynamics and Pharmacokinetics 2008, 8, 153.

  • 17.

    Kim, H.; Hong, J.; Park, M.; Kang, J.; Lee, M. Biomed. Chromatogr. 2001, 15, 539.

  • 18.

    Gaginella, T. S. Handbook of Methods in Gastrointestinal Pharmacology CRC Press, Informa group, London, 1995.

  • 19.

    Bailey, L. E.; Ong, S. D. J. Pharmacol. Methods 1978, 1, 171.

  • 20.

    EMEA, European Medicines Agency (EMA), Committee for Medicinal Products for Human Use (CHMP) 2012.

  • 21.

    Custodio, J. M.; Wu, C.-Y.; Benet, L. Z. Adv. Drug Deliv. Rev. 2008, 60, 717.

  • 22.

    Wu, S. T.; Chang, Y. P.; Gee, W. L.; Benet, L. Z.; Lin, E. T. J. Chromatogr. B Biomed. Sci. Appl. 1997, 692, 133.

  • 23.

    Christie, W. W. Advances in Lipid Methodology vol. 4, Woodhead Publishing Limited, UK, 2012, 239.

  • 24.

    Srikanth, M.; Ram, B. J.; Sunil, S.; Rao, N. S.; Murthy, K. R. Journal of Scientific and industrial Research 2012, 71, 120.

  • 25.

    Kupiec, T. Int. J. Pharm. Compd. 2004, 8, 223.

  • 26.

    McMaster, M. HPLC: a practical user's guide John Wiley & Sons, Hoboken, New Jersey, 2007.

  • 27.

    Zhou, M. Regulated Bioanalytical Laboratories: Technical and Regulatory Aspects from Global Perspectives John Wiley & Sons, Hoboken, New Jersey, 2011.

    • Crossref
    • Search Google Scholar
    • Export Citation

If the inline PDF is not rendering correctly, you can download the PDF file here.

  • 1.

    Salman, S. A.; Sulaiman, S. A.; Ismail, Z.; Gan, S. H. Toxicol. Mech. Methods 2010, 20, 137.

  • 2.

    Cole, S. W.; Sood, A. K. Clin. Cancer Res. 2012, 18, 1201.

  • 3.

    Priviero, F. B.; Teixeira, C. E.; Claudino, M. A.; De Nucci, G.; Zanesco, A.; Antunes, E. Eur. J. Pharmacol. 2007, 571, 189.

  • 4.

    Hebb, A. R.; Godwin, T. F.; Gunn, R. W. Can. Med. Assoc. J. 1968, 98, 246.

  • 5.

    Bruns, L. A.; Canter, C. E. Paediatr Drugs 2002, 4, 771.

  • 6.

    WHO, , World health organization model list of essential medicines: 17th list. Available at http://apps.who.int/iris/bitstream/10665/70640/1/a95053_eng.pdf, 2011. Access date: 1 October 2015.

  • 7.

    Hackett, L.; Dusci, L. Clin. Toxicol. 1979, 15, 63.

  • 8.

    Semple, H. A.; Xia, F. J. Chromatogr. B Biomed. Sci. Appl. 1994, 655, 293.

  • 9.

    Rekhi, G. S.; Jambhekar, S. S.; Souney, P. F.; Williams, D. A. J. Pharm. Biomed. Anal. 1995, 13, 1499.

  • 10.

    Braza, A. J.; Modamio, P.; Mariño, E. L. J. Chromatogr. B Biomed. Sci. Appl. 2000, 738, 225.

  • 11.

    Zhanga, J.; Dinga, L.; Wenb, A.; Wua, F.; Suna, L.; Yangb, L. Asian J. Pharm. Sci. 2009, 4, 169.

  • 12.

    Li, S.; Liu, G.; Jia, J.; Liu, Y.; Pan, C.; Yu, C.; Cai, Y.; Ren, J. J. Chromatogr. B 2007, 847, 174.

  • 13.

    Umezawa, H.; Lee, X. P.; Arima, Y.; Hasegawa, C.; Izawa, H.; Kumazawa, T.; Sato, K. Biomed. Chromatogr. 2008, 22, 702.

  • 14.

    Delamoye, M.; Duverneuil, C.; Paraire, F.; de Mazancourt, P.; Alvarez, J.-C. Forensic Sci. Int. 2004, 141, 23.

  • 15.

    El-Saharty, Y. J. Pharm. Biomed. Anal. 2003, 33, 699.

  • 16.

    Xiao, S.; Wei, G.; Guo, H.; Liu, H.; Liu, C. Asian Journal of Pharmacodynamics and Pharmacokinetics 2008, 8, 153.

  • 17.

    Kim, H.; Hong, J.; Park, M.; Kang, J.; Lee, M. Biomed. Chromatogr. 2001, 15, 539.

  • 18.

    Gaginella, T. S. Handbook of Methods in Gastrointestinal Pharmacology CRC Press, Informa group, London, 1995.

  • 19.

    Bailey, L. E.; Ong, S. D. J. Pharmacol. Methods 1978, 1, 171.

  • 20.

    EMEA, European Medicines Agency (EMA), Committee for Medicinal Products for Human Use (CHMP) 2012.

  • 21.

    Custodio, J. M.; Wu, C.-Y.; Benet, L. Z. Adv. Drug Deliv. Rev. 2008, 60, 717.

  • 22.

    Wu, S. T.; Chang, Y. P.; Gee, W. L.; Benet, L. Z.; Lin, E. T. J. Chromatogr. B Biomed. Sci. Appl. 1997, 692, 133.

  • 23.

    Christie, W. W. Advances in Lipid Methodology vol. 4, Woodhead Publishing Limited, UK, 2012, 239.

  • 24.

    Srikanth, M.; Ram, B. J.; Sunil, S.; Rao, N. S.; Murthy, K. R. Journal of Scientific and industrial Research 2012, 71, 120.

  • 25.

    Kupiec, T. Int. J. Pharm. Compd. 2004, 8, 223.

  • 26.

    McMaster, M. HPLC: a practical user's guide John Wiley & Sons, Hoboken, New Jersey, 2007.

  • 27.

    Zhou, M. Regulated Bioanalytical Laboratories: Technical and Regulatory Aspects from Global Perspectives John Wiley & Sons, Hoboken, New Jersey, 2011.

    • Crossref
    • Search Google Scholar
    • Export Citation

Monthly Content Usage

Abstract Views Full Text Views PDF Downloads
Jun 2020 0 19 5
Jul 2020 0 29 2
Aug 2020 0 34 6
Sep 2020 0 28 3
Oct 2020 0 39 12
Nov 2020 0 20 4
Dec 2020 0 0 0