Development and Validation of an Isocratic HPLC Method for Simultaneous Determination of Quaternary Mixtures of Antihypertensive Drugs in Pharmaceutical Formulations

Original Acta Chromatographica 29(201 7)1, 95 –110 Research Paper DOI: 10.1556/1326.2017.29.1.9

J.F.F. A NDERSON 1, M.C.G. G ERLIN 1, R.A. S VERSUT 1, L.C.S. O LIVEIRA 2, A.K. S INGH 3, M.S. A MARAL 1,4 , AND N.M. K ASSAB 1,*

1Programa de Pós-Graduação em Farmácia, Centro de Ciências Biológicas e da Saúde, Universidade Federal de Mato Grosso do Sul, Brazil 2Instituto de Química, Universidade Federal de Mato Grosso do Sul, Brazil 3Departamento de Farmácia, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, 05508-900, São Paulo, SP, Brazil 4Instituto de Física, Universidade Federal de Mato Grosso do Sul, Brazil *E-mail: [email protected]

Summary. The objective of this study was to develop and validate an assay method for simultaneous determination of atenolol, furosemide, losartan, and spironolactone in pharmaceutical formulations. A reverse-phase high-performance liquid chromatography procedure was developed, using a Kinetex ® C-18 column (100 mm × 4.6 mm, 2.6 µm). The mobile phase was composed of methanol–water (75:25 v/v, pH 3.0, adjusted with phosphoric acid), with a flow rate of 0.4 mL min −1. All drugs were separated in less than 5 min. The method was validated according to International Conference on Harmoniza- tion (ICH) and Association of Official Analytical Chemists (AOAC) guidelines. The method showed linearity in a concentration range of 0.75–12.0 µg mL −1 for atenolol (r = 0.9995), 0.30–12.00 µg mL −1 for furosemide ( r = 0.9997), 0.45–12.00 µg mL −1 for losartan ( r = 0.9995), and 0.45–12.0 µg mL −1 for spironolactone ( r = 0.9999). The method also showed repeatability and precision. The three-day average intra-day precisions were 101.35 ± 0.74% for atenolol, 95.84 ± 1.44% for furosemide, 98.90 ± 1.16% for losartan, and 97.19 ± 0.18% for spironolactone. Similarly, the inter-day precisions were 101.34 ± 0.72% for atenolol, 95.84 ± 0.1.50% for furosemide, 98.90 ± 1.17% for losartan, and 97.19 ± 0.83% for spironolactone. The method accuracy was also tested and validated — in this case, the average recovery values were 100.18 ± 1.20% for atenolol, 99.83 ± 1.54% for furosemide, 100.07 ± 0.95% for losartan, and 99.94 ± 0.93% for spironolactone. Finally, the method was successfully applied in the simultaneous determination of atenolol, furosemide, losartan, and spironolactone in magisterial formulas, as well as in commercial pharmaceutical formulations.

Key Words: HPLC–DAD, antihypertensive, quality control, simultaneous determination, quantitative

This is an open -access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium for non - commercial purposes, provided the original author and source are credited. First published online: April 10, 2016 ISSN 2083-5736 © The Author(s)

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Introduction

Hypertension is a main risk factor for cardiovascular and kidney diseases and is among the leading causes of mortality in Brazil and worldwide [1]. A very common disorder, hypertension, if neglected, can lead to coronary thrombosis, stroke, and kidney failure [2, 3]. Atenolol (Fig. 1) is a β-adrenergic receptor antagonist with cardioselec- tive activity and is widely used by hypertensive patients, especially in sus- pected angina or heart failure [3]. Its clinical use can be attributed to its car- dioprotective effect, and its effectiveness as an antihypertensive is well described when used in combination with a diuretic for elderly patients with isolated hypertension [2].

Fig. 1 . Chemical structures of hypertensive drugs used in the method validation

Furosemide (Fig. 1) is a loop diuretic, able to cause elimination of 15–25% of filtered Na +[3], and is widely used in the treatment of hyperten-

Unauthenticated | Downloaded 10/02/21 06:31 PM UTC Isocratic HPLC Method 97 sion [2]. Compared to other diuretics such as the thiazides, furosemide causes smaller disturbances in the lipid profile [2, 4]. Losartan (Fig. 1) is an angiotensin II receptor antagonist and has a smooth-muscle relaxing action. The vasodilation activity of losartan increases renal excretion of water and salt, reduces plasma volume, and de- creases cellular hypertrophy [2]. It has a satisfactory hypotensive effect and is indicated mainly for young persons, diabetics, or patients with heart failure [2, 3]. Spironolactone (Fig. 1) is a diuretic, although it has very limited effectiveness when used alone, being responsible for reabsorption of only 2% of the Na + filtered [2, 3]. However, spironolactone has an accentuated antihypertensive effect when combined with other diuretics, such as loop diuretics. This combination increases mobilization of body fluid, while causing less disturbance in K + homeostasis. When combined with conventional therapy, spironolactone substantially reduces morbidity and mortality in patients with heart failure [2, 5]. Combined-dose formulations have several advantages in the treatment of hypertensive patients, such as increased antihypertensive efficacy result- ing from the combination of drugs that act by different mechanisms, reduc- tion of adverse effects due to the use of lower doses, simplification, and im- provement of treatment adherence [1, 6]. In the case of antihypertensive therapy, adherence to the treatment is one of the major challenges in the treatment of hypertensive patients. Stud- ies show that patient compliance with the treatment is inversely propor- tional to the number of units taken daily and/or the number of daily doses. Nearly 2/3 of hypertensive patients require the combined dosage form for better compliance with the treatment [1]. The compounding pharmacy provides tailor-made capsules in a form that allows a custom drug dosage. In addition to the lower cost of the compounded dosage form for better adjustment of the dose, these formulations play an important role when no commercial combined dosage forms are available in the market [7]. Although compounding is often preferred, since it meets the needs of the individual patient, the Brazilian Society of Cardiol- ogy (SBC) does not recommend compounding, due to a lack of information on quality standards [8], bio-equivalence, and/or chemical interactions among the compounded drugs [9]. For these reasons, quality control of pharmaceutical products is indis- pensable, not only to ensure the effectiveness of production processes but also and primarily to ensure compliance with qualitative and quantitative standards that ensure the effectiveness and safety of pharmaceuticals.

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Table I. Analytical methods described in the literature for the analysis of atenolol, furosemide, losartan, and spironolactone

Method Reference Drug Type of sample

Atenolol, atorvastatin, Commercial [10] acetylsalicylic acid, losartan formulation Atenolol, sotalol, diacetolol, carteolol, nadolol, pindolol, [11] acebutolol, metoprolol, celiprolol, Human plasma oxprenolol, labetalol, propranolol, tertatolol, betaxolol Raw material and [12] Spironolactone, torsemide tablet High-performance Combinations of liquid chromatography hydrochlorothiazide with: atenolol, Pharmaceutical [13] (HPLC) amlodipine, candesartan, moexipril, formulation valsartan [14] Carvedilol, losartan Plasma and urine Pharmaceutical [15] Hydrochlorothiazide, losartan formulation Atenolol, amlodipine, atorvastatin, Pharmaceutical [16] enalapril, hydrochlorothiazide, formulation lisinopril, losartan [17] Spironolactone, metolazone Tablet and urine Spironolactone, furosemide, Pharmaceutical Densitometry [18] hydrochlorothiazide, triamterene formulation Capillary Pharmaceutical [19] Atenolol, amiloride electrophoresis formulation Ultraviolet spectrophotometry [20] Hydrochlorothiazide, losartan Tablet (UV) Pharmaceutical Irbesartan, losartan, olmesartan, [21] formulation and valsartan Thin-layer human plasma chromatography Amlodipine, hydrochlorothiazide, Pharmaceutical (HPTLC) [22] losartan formulation [23] Atorvastatin, losartan Tablet

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Table I. (continued)

Method Reference Drug Type of sample Pharmaceutical Liquid chromatography [24] Tranexamic acid, losartan formulation and (LC) serum Acetylsalicylic acid, atenolol, Liquid chromatography bisoprolol, chlorthalidone, enalapril coupled to mass [25] and its active metabolites, Plasma spectrometry (LC– fluvastatin, hydrochlorothiazide, MS/MS) valsartan [26] Atenolol, nifedipine Plasma

Several analytical methods have been described for the analysis of atenolol, furosemide, losartan, and spironolactone, in isolation or in combined dosage form (Table I). However, we are unaware of a method for simultaneous determination of all four drugs in a single pharmaceutical formulation. The objective of this study was to develop a single chromatographic method for simultaneous determination of all four drugs in compounded capsule form.

Experimental

Materials and Reagents

The raw materials were purchased from distributors licensed by the Na- tional Health Surveillance Agency (ANVISA, Brazil). The stated purity levels were: atenolol 98.86%, furosemide 100.75%, losartan 99.12%, and spironolactone 99.4%. The placebo, consisting of 1% silicon dioxide, 59% micro- crystalline cellulose, and 40% corn starch was also purchased from a local market. Three samples were acquired from each of three different local compounding pharmacies. In all cases, raw materials were used as reference standards without further purification. The samples were kept protected from light. High-performance liquid chromatography (HPLC)-grade methanol (Vetec Química Fina, Brazil), analytical-grade phosphoric acid (Synth, Brazil), and ultrapure water obtained with a Millipore Direct-Q3 ® water purification system were used in all analyses.

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Instrumentation and Chromatographic Conditions

The HPLC analyses were conducted in a system consisting of a Dionex ® HPLC system, model Ultimate 3000 (Thermo Fisher Scientific, USA), equipped with an ultraviolet (UV) diode array detector (DAD) (Ultimate 3000 RS) and binary pumps (Ultimate 3000). The chromatographic data were processed with the software Chromeleon ® 7.1. The injection volume was set at 20 µL. Experimental conditions were optimized on a Kinetex® C-18 column (100 mm × 4.6 mm, 2.6 µm; Phenomenex, USA). The separations were obtained in isocratic mode, using a mobile phase consisting of methanol–water (75:25 v/v, pH 3.0, adjusted with phosphoric acid), with a flow rate of 0.4 mL min −1, and detection was done at 225 nm for atenolol and 240 nm for furosemide, losartan, and spironolactone. All analyses were conducted at room temperature (24 ± 2 °C).

Standard Stock Solutions and Calibration Curves

Stock solutions of the reference standards were prepared in the mobile phase and used on the same day. The stock solutions contained 100 µg mL −1 atenolol, 40 µg mL −1 furosemide, 100 µg mL −1 losartan, or 50 µg mL −1 spironolactone. Under the experimental conditions described, each compound’s linear calibration curve – chromatographic peak areas versus drug concentration – was constructed by diluting the stock solution (to at least eleven concentration levels) in the mobile phase. On the calibration curves, the lowest concentration in linear range was obtained by injecting successive diluted solutions from a standard solution at a concentration of 1.5 µg mL −1. This way, the lowest sample quantity was determined with good precision and accuracy. By definition, these lowest concentrations obtained are the limit of quantification (LOQ) for each drug of the proposed method. Fur- thermore, all chromatographic determinations were performed in triplicate and at room temperature (24 ± 2 °C).

Tablet Solution Preparation

Capsules containing 50 mg atenolol, 20 mg furosemide, 50 mg losartan, or 25 mg spironolactone were weighed individually, and the contents of each capsule were then removed and weighed. The amounts diluted in the mo-

Unauthenticated | Downloaded 10/02/21 06:31 PM UTC Isocratic HPLC Method 101 bile phase, based on the mean weight of the capsule contents, were 10 µg mL −1 atenolol, 4 µg mL −1 furosemide, 10 µg mL −1 losartan, and 5 µg mL −1 spironolactone.

Results and Discussion

Method Optimization

The optimum mobile phase for simultaneous and quantitative determination of the four drugs was determined according to previous published results (Table I) and after tests of several solvent mixtures (different propor- tions), as well as the use of organic modification.

Table II . System suitability parameters of the proposed HPLC–DAD method for determination of antihypertensive drugs

Parameter Obtained value a Reference value b

R1,2 = 2.23

Resolution ( R) R2,3 = 2.99 R > 2.0 R3,4 = 2.71

T1 = 1.08

T2 = 1.15 Accepted T values from Tailing factor ( T) T3 = 1.14 (0.9–1.2)

T4 = 1.16 K1 = 1.34

K2 = 1.89 Accepted K values from Capacity factor ( K) K3 = 2.64 (1–10)

K4 = 3.39

N1 = 2,470 Number of theoretical plates N2 = 3,266 Accepted N values from

(N) N3 = 4,164 (>2000)

N4 = 5,170

HETP 1 = 4.05 × 10 −3 The lower the HETP HETP (length of column in HETP 2 = 3.06 × 10 −3 value, the higher the cm/ N) HETP 3 = 2.40 × 10 −3 column efficiency

HETP 4 = 1.93 × 10 −3 a1, 2, 3, and 4 indicate atenolol, furosemide, losartan, and spironolactone, respectively. bAccording to Food and Drug Administration (FDA) Reviewer Guidance of Valida- tion of Chromatographic Methods [29].

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The methanol–water (80:20, v/v) mixture was the first mobile phase tested. Unfortunately, it was not possible to obtain complete and efficient peak separation. Considering drug ionization [27, 28], the pH of the methanol–water (80:20, v/v) mixture was adjusted to 3.0, using phosphoric acid. This improved the peak separation of the drugs, although the resolution of atenolol and furosemide continued to be insufficient. Finally, the methanol– water (75:25, v/v, pH 3.0) mixture showed symmetrical peaks (tailing factor between 0.90 and 1.20), resolution >2.0, theoretical plates >2000, and in the following order of elution: atenolol, furosemide, losartan, and spironolactone, with retention times ( tR) of 2.11, 2.58, 3.23, and 3.88 min, respectively. Table II shows all system suitability parameters of the proposed and optimized HPLC–DAD method for simultaneous determination of the four antihypertensive drugs.

Validation

The proposed HPLC–DAD method was validated according to the guidelines of the International Conference on Harmonization (ICH) and the Asso- ciation of Official Analytical Chemists (AOAC). In addition to the system suitability, the selectivity, linearity, precision, accuracy, robustness, limit of quantitation, and limit of detection were evaluated [30–32].

Table III. Validation data for the determination of atenolol, furosemide, losartan, and spironolactone using the proposed HPLC–DAD method

Atenolol Furosemide Losartan Spironolactone Parameters a (at 225 nm) (at 240 nm) (at 240 nm) (at 240 nm) Concentration range 0.75–12.00 0.30–12.00 0.45–12.00 0.45–12.00 (µg mL −1) Correlation coefficient ( r) 0.9995 0.9997 0.9995 0.9999 Slope (mL µg−1) 1.9433 3.5176 2.1472 2.7421 Intercept 0.5744 0.5750 0.9123 -0.1150 − Sa (mL µg 1) 0.1406 0.1807 0.1543 0.0648

Sb 0.0217 0.0291 0.0249 0.0100 LOD ( µg mL −1) 0.24 0.17 0.24 0.08 LOQ ( µg mL −1)a 0.75 (0.72) 0.30 (0.51) 0.45 (0.72) 0.45 (0.24)

LOD: limit of detection, LOQ: limit of quantitation, Sa: standard deviation of y- intercept, Sb: standard deviation of slope. a The theoretical results obtained by equation 10 × Sa / slope are in parentheses.

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Fig. 2 . Chromatograms for 225 nm and 240 nm wavelength detection: (A) placebo solution and (B) commercial sample solution for atenolol (ATN), furosemide (FRS), losartan (LST), and spironolactone (SPR). Numbers above the peaks indicate the retention times ( tR)

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Linearity

The concentration ranges for each calibration curve are listed in Table III . The slopes, intercepts, and correlation coefficients obtained by the linear least-squares regression treatment of the results are also given. The high values of the correlation coefficients ( r > 0.998) indicate the good linearity of the calibration graphs.

Selectivity and Specificity

The selectivity was evaluated by analyzing a solution containing all the excipients (1% silicon dioxide, 59% microcrystallized cellulose, and 40% corn starch) used in the compounded capsules. The results obtained with the placebo were compared to those obtained with the sample solutions. The mixture of excipients used in the compounded combined-dose capsule did not affect the chromatographic analyses (Fig. 2) .

Repeatability and Precision

The precision of the method was evaluated with respect to intra-day precision (repeatability) and inter-day precision (intermediate precision). The relative standard deviation (RSD) values, in all cases, were less than 1.5% for repeatability and intermediate precision (Table IV) . These results show that the proposed method has adequate precision in simultaneous determination of these drugs in pharmaceutical formulations.

Table IV . Data for precision obtained in the inter- and intra-day simultaneous analyses of four drugs

Theoretical Experimental concentrations ± RSD c (%) concentra- Drug a tion Repeatability Intermediate (µg mL −1) Day 1 Day 2 Day 3 precision b Atenolol 10 102.18 ± 0.45 101.08 ± 0.26 100.78 ± 0.71 101.34 ± 0.72 Furosemide 4 96.58 ± 0.22 96.75 ± 0.58 94.18 ± 0.55 95.84 ± 1.50 Losartan 10 100.05 ± 0.73 98.93 ± 0.30 97.73 ± 0.29 98.90 ± 1.17 Spironolactone 5 97.98 ± 0.36 97.23 ± 0.29 96.36 ± 0.23 97.19 ± 0.83 aMean of 6 determinations. bMean of 3 determinations performed on three different days. cRelative standard deviation.

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Accuracy

The accuracy of the method was determined by recovery studies using the standard addition method, in accordance with AOAC Guidelines [33]. The recovery was analyzed by adding known amounts of standard solutions to the sample solutions, followed by analysis using the proposed method. The mean recovery values (Table V) are in accordance with the acceptable limits (100 ± 2%) and indicate the accuracy of the method.

Table V . Data for recovery in simultaneous determination of four drugs by the proposed method Final experi- Recovery Final theoretical mental con- concentration b Drug centration a Results Mean ± RSD (µg mL −1) (%) (%) (µg mL −1) 2.00 1.99 99.29 Atenolol 4.00 3.99 99.71 100.18 ± 1.20 6.00 6.09 101.54 2.00 1.97 98.26 Furosemide 4.00 3.99 99.87 99.83 ± 1.54 6.00 6.08 101.35 2.00 1.98 99.00 Losartan 4.00 4.02 100.45 100.07 ± 0.95 6.00 6.05 100.78 2.00 1.98 99.20 Spironolactone 4.00 4.04 100.98 99.94 ± 0.93 6.00 5.98 99.64

aMean of 3 determinations. bRelative standard deviation.

Robustness

Robustness was evaluated by deliberate subtle changes in the chromatographic conditions, and their effects on the peak area, retention time ( tR), and asymmetry factor (AS) were monitored. The factors (chromatographic conditions) selected for the analysis of robustness were the proportion of methanol in the mobile phase, pH of the mobile phase, and flow rate. Each factor was parsed into three levels (0, −1, and +1). Level 0 refers to the standard conditions employed in the method. From this level, the chroma-

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tographic conditions were modified to a higher level (+1) or a lower level (−1). A single factor was changed at a time, while others remained at level 0. The results obtained in the robustness test indicated that small changes in the chromatographic conditions had insignificant impacts on the peak areas, retention time, and peak asymmetry in the analyses of all four drugs (Table VI) . In all cases, adequate separation of all four drugs for quantitative determination was obtained.

Table VI. Results of the robustness test with proposed chromatographic method

Chromatographic conditions Parameters Peak ar- Retention Pharmaceuticals Factor a Level Asymmetry ea time (min) A: % methanol in mobile phase (v/v) 74 −1 21.13 2.14 1.33 Atenolol 75 0 19.99 2.11 1.33 76 1 20.51 2.14 1.27 74 −1 15.48 2.60 1.14 Furosemide 75 0 15.13 2.58 1.16 76 1 15.39 2.55 1.17 74 −1 22.78 3.31 1.14 Losartan 75 0 22.41 3.23 1.15 76 1 22.83 3.12 1.14 74 −1 13.24 4.04 1.17 Spironolactone 75 0 12.96 3.88 1.19 76 1 13.27 3.74 1.13 B: Mobile phase pH 2.99 −1 21.39 2.14 1.27 Atenolol 3.00 0 19.99 2.11 1.33 3.01 1 21.26 2.13 1.24 2.99 −1 16.30 2.58 1.16 Furosemide 3.00 0 15.13 2.58 1.16 3.01 1 16.39 2.58 1.16 2.99 −1 24.05 3.21 1.14 Losartan 3.00 0 22.41 3.23 1.15 3.01 1 23.95 3.23 1.15 2.99 −1 13.99 3.88 1.15 Spironolactone 3.00 0 12.96 3.88 1.19 3.01 1 13.97 3.89 1.16

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Table VI. (continued)

Chromatographic conditions Parameters Peak ar- Retention Factor a Level Asymmetry Pharmaceuticals ea time (min) C: Flow rate (mL min −1) 0.39 −1 20.91 2.23 1.37 Atenolol 0.40 0 19.99 2.11 1.33 0.41 1 18.84 2.13 1.25 0.39 −1 14.95 2.62 1.24 Furosemide 0.40 0 15.13 2.58 1.16 0.41 1 12.35 2.49 1.19 0.39 −1 23.39 3.25 1.19 Losartan 0.40 0 22.41 3.23 1.15 0.41 1 20.04 3.08 1.14 0.39 −1 13.20 3.95 1.19 Spironolactone 0.40 0 12.96 3.88 1.19 0.41 1 12.01 3.75 1.13

a Factors (A, B, C) were altered at three levels (−1, 0, 1).

Limit of Detection and Limit of Quantification

The limit of detection (LOD) was calculated based on the standard deviation and the angular coefficient (slope) of the calibration curve. The LOD was theoretically obtained using the equation 3.3 σ/s, where σ represents the standard deviation of the y-intercept and s is the slope of the corre- sponding calibration curve. As described in Experimental section, the LOQ was determined experimentally instead of calculation by equation 10 σ/s. Table III shows the LOD and LOQ values for all drugs.

Applicability of the Proposed Method

Commercial samples containing 50 mg atenolol, 20 mg furosemide, 50 mg losartan, and 25 mg spironolactone were acquired randomly in the local market and analyzed with the proposed HPLC–DAD method. The results showed that the chromatographic method is suitable for simultaneous determination of these four drugs in compounded formulations and can be

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Table VII. Analysis of compounded commercial formulations using the proposed HPLC– DAD method

Declared Compounded Amount found ± Drug concentration formula a RSD (%) b (µg mL −1) Atenolol 10.00 101.86 ± 1.25

Furosemide 4.00 96.91 ± 1.41 Sample 1 Losartan 10.00 97.51 ± 0.28 Spironolactone 5.00 96.36 ± 1.47 Atenolol 10.00 105.68 ± 0.31 Furosemide 4.00 100.37 ± 0.91 Sample 2 Losartan 10.00 96.96 ± 0.44 Spironolactone 5.00 100.61 ± 0.51 Atenolol 10.00 96.78 ± 0.94 Furosemide 4.00 92.23 ± 1.82 Sample 3 Losartan 10.00 99.52 ± 0.66 Spironolactone 5.00 92.72 ± 0.76

aCommercial sample (Compounded formula 1, 2, 3 available in capsules). bMean of three determinations.

Conclusions

An ultrafast and isocratic HPLC–DAD method was developed and validated for simultaneous determination of atenolol, furosemide, losartan, and spironolactone in combined-dose compounded formulations. In addition to cost-effectiveness and faster analysis, the proposed method showed specificity, precision, accuracy, sensitivity, and robustness in the analyses of all drugs in the compounded formulations. The major advantage of the proposed method is its efficiency, wherein a combined dosage of any combination of the drugs or in their isolated form

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