Fluorescamine-based fluorophore for spectrofluorimetric determination of heptaminol in human plasma; application to spiked human plasma

Journal Pre-proof Fluorescamine‐based fluorophore for spectrofluorimetric determination of heptaminol in human plasma; application to spiked human plasma Mahmoud A. Omar, Dalia M. Nagy, Monica E. Halim PII:

S1386-1425(19)31101-1

DOI:

https://doi.org/10.1016/j.saa.2019.117711

Reference:

SAA 117711

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received Date: 6 May 2019 Revised Date:

17 October 2019

Accepted Date: 24 October 2019

Please cite this article as: M.A. Omar, D.M. Nagy, M.E. Halim, Fluorescamine‐based fluorophore for spectrofluorimetric determination of heptaminol in human plasma; application to spiked human plasma, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2019), doi: https:// doi.org/10.1016/j.saa.2019.117711. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

800 Drug emmision Drug excitation Blank emmision Blank excitation

RFI

600

400

200

0 300

350

400

450

500

550

600

Wavelength (nm)

Excitation and emission spectra of the products of the reaction between heptaminol and fluorescamine.

Fluorescamine based fluorophore for spectrofluorimetric determination of Heptaminol in human plasma; Application to spiked human plasma Mahmoud A. Omar*, Dalia M. Nagy and Monica E. Halim. Analytical Chemistry Department, Faculty of Pharmacy, Minia University, Minia, Egypt. Abstract An innovative approach to determine heptaminol spectrofluorimetrically was developed, determining the optimum conditions needed, then validated for determination of heptaminol in its pure form, its tablets and in spiked human plasma. The presented method is based on the reaction between fluorescamine reagent with the primary amine group found in heptaminol, using a borate buffer at pH 9.0 that yields a highly fluorescent product, fluorescence was measured at 471 nm after excitation at 393 nm. The linearity of the constructed calibration curve was (75 – 850 ng/ml) with LOD and LOQ values 23.85 and 72.29 ng/ml respectively. The method was validated following the International Council for Harmonisation (ICH) guidelines indicating good accuracy and precision. Finally, the presented approach was adapted for in vitro study of heptaminol in spiked human plasma with a mean percentage recovery 100.52 ± 1.19% as well as in its tablets with a mean percentage recovery 99.47 ± 1.25 %. Keywords: heptaminol, fluorescamine, spectrofluorimetric determination, spiked plasma. 1. Introduction Heptaminol (6-Hydroxy-6-methyl-2-heptylamine Hydrochloride) is an amino alcohol drug. (Figure 1) It shows a positive inotropic action on the cardiovascular system, thus, increasing the coronary blood flow along with slight peripheral vasoconstriction. This makes it a myocardial stimulant that is the most common choice to treat orthostatic hypotension. It is also used for catecholamine weaning in septic shock. [1-4] Various methods were published for determining heptaminol in both biological fluids and pharmaceutical preparations, they included spectrophotometric [5-8], spectrofluorometric [8-10], capillary electrophoretic [11] and chromatographic [12-21] methods.

In the presented study, a new spectrofluorimetric approach for determining heptaminol, in both its pure authentic form or its tablets, was discussed in order to optimize and validate all the variables of the chemical reaction between the cited drug and fluorescamine. Owing to the absence of native fluorescence of heptaminol, the use of a fluorogenic reagent, as fluorescamine, makes it a good method for its analysis spectrofluorimetrically. The designed approach can be considered an improved method over other previously reported spectrofluorometric methods due to its feasibility and expeditiousness, as neither heating nor extraction is needed with merely 10 minutes required to get a complete reaction while being both sensitive and accurate for determination of heptaminol in its pharmaceutical tablets during quality control analysis. 2. Experimental 2.1. Apparatus In order to collect the data, the apparatus used was the LS 45 spectrofluorometer (Perkin-Elmer, Coventry, UK) that consisted of a xenon lamp (150-watt) together with a quartz cuvette with a 1 cm path length. Excitation and emission monochromators included in the spectrometer were set to have 10 nm as an opening width. To this spectrometer a PC computer where FL WINLABTM software was installed was linked. Milwaukee SM 101 PH meter (Portugal) was among the other instruments used as well as the digital analytical balance (AG 29, Mettler Toledo, Glattbrugg, Switzerland).

2.2. Reagents and materials Heptaminol was gifted by Pharco. for pharmaceutical industries (Cairo, Egypt) Corasore® tablet (containing 150 mg of heptaminol per tablet) manufactured by Amoun pharmaceutical Co was analyzed. Chemicals as boric acid, sodium hydroxide, acetonitrile, ethanol, methanol, acetone, and hydrochloric acid were purchased from EL-Nasr Chemical Co. (Cairo, Egypt). Using acetone as a solvent 0.02 %w/v fluorescamine (Alfaaesar, ThermoFischer, Germany) was prepared, it was stable when stored at 4°C for about a week. Using borate buffer (prepared by blending 0.1 M solutions of both sodium hydroxide and boric acid in varied volumes) the pH range from 7.7 to 12.0 was rendered.

2.3. Standard solution preparation Using distilled water as a diluting solvent, accurately 10.0 mg of the cited drug was dissolved in a 100 ml volumetric flask from which 5 ml was further diluted to 100 ml to obtain a stock solution that has 5 µg/ml as the final concentration from which various working solutions were prepared through making additionally dilutions using the same diluting solvent. The working solutions had concentrations ranging from 0.75 to 8.5 µg/ml, their stability lasted for a couple of weeks when refrigerated at 4°C.

2.4. General analytical procedure 0.7 ml of borate buffer at pH 9.0 and 1.0 ml of fluorescamine (0.02 %w/v using acetone) were added to 1.0 ml of working solutions that has a concentration ranging from 0.75 to 8.5 µg/ml using a series of calibrated flasks. The volumes were then completed to the total volume of 10.0 ml using ethanol as a diluent and left standing for 10 minutes to get a complete reaction. The fluorescence intensity of the formed product was measured at 471 nm after being excited at 393 nm. By executing the same procedure, but omitting the addition of heptaminol, a blank was obtained simultaneously.

2.5. Determination of the cited drug in its tablet A specific amount of the powder of twenty tablets of Corasore® corresponding to 10.0 mg of the active constituent (heptaminol) was weighed to be dissolved, then filtered into a 100.0 ml calibrated flask using distilled water as a diluting agent. This filtrate was additionally diluted using distilled water, giving working solutions with concentration ranging from 0.75 to 8.5 µg/ml, to which the same steps of the general method were executed.

2.6. Determination of the stoichiometry of the reaction The stoichiometric ratio between heptaminol and fluorescamine was figured out using Job’s method through using equimolar concentrations of both heptaminol and the fluorogenic reagent (4.0 X 10-4 M). Complementary volumes of the two solutions were added, into a series of 10.0 ml calibrated flasks, till their total volume was one meal on which the same routine procedure was executed. For each flask, the resulted product’s fluorescence was measured. A graph was

then plotted between the product’s relative fluorescence intensity (RFI) versus their parallel heptaminol mole fractions.

2.7 Procedures for spiked human plasma A five ml heptaminol-free blood sample, gathered from a healthy human volunteer, was taken into a heparinized tube to be centrifuged at 4000 round per minute for half an hour. One ml of the collected supernatant (plasma free of the drug) was spiked with 1.0 ml of heptaminol working solutions with a concentration ranging from 7.5 to 85 µg/ml in a 10.0 ml stoppered calibrated tube. To this combination, two ml of acetonitrile, that acts as a protein precipitating agent, was added then diluted to 10.0 ml using distilled water to get solutions with 0.75 to 8.5 µg/ml as their final concentrations. The final mixture was then centrifuged for a further 20 minutes at 4000 rpm until we get a plasma spiked with the drug (supernatant) on which the general method was executed on 1.0 ml of it. The same procedure was followed on a heptaminolfree plasma sample in order to obtain a blank reading.

3. Results and discussion Fluorescamine is significantly used for detection of proteins, amino acids, peptides or proteolytic enzymes’ activity.[22-26] Upon the reaction of fluorescamine with a primary amine moiety as the one present in the studied drug (heptaminol), it gives a product that is highly fluorescent showing emission at 471 nm subsequent to being excited at 393 nm. (Figure 2) The reaction’s proposed pathway was presumed in figure 3 showing a nucleophilic attack between fluorescamine and the primary group of heptaminol giving a pyrrolone form which is highly conjugated and responsible for the produced fluorescence. 3.1. Experimental factors optimization For revealing the effect of each factor on either the reaction’s product fluorescence or the stability, each factor was independently varied while others were kept constant. The factors that needed optimization included the pH, volume of the buffer, volume of fluorescamine reagent, as well as heating temperature and time. The selection of the most optimum value for each factor was the value that gives the highest RFI.

3.1.1. pH’s effect

To get a complete reaction with higher fluorescence, the pH needs to be rendered alkaline, which was done using a borate buffer that covered a wide range of pH units from (7.7 to 12.0). The fluorescence of the formed product reached its highest value within the pH range of 8.8 to 9.5, thus making pH 9.0 the most appropriate pH needed for the reaction. (Figure 4)

3.1.2. The volume of buffer’s effect The complete absence of a buffer showed a notable decrease in the RFI values, so varied volumes till 2.5 ml of borate buffer at pH 9.0 was used to figure out the volume giving the peak fluorescence. The highest RFI was observed at 0.5 ml and remained steady till 1.0 ml above which a decline in the fluorescence occurred. This makes 0.7 ml of borate buffer pH 9.0 the most excellent conditions to be used. (Figure 4)

3.1.3. The volume of fluorogenic reagent ’s effect Varied volumes of 0.02% w/v Fluorescamine (0.2 to 1.6 ml) were utilized showing their effect on the product’s fluorescence. With the increase in the volume of the reagent, the fluorescence of the formed product increased until a steady state was reached at volumes (0.8 to 1.4 ml), a further increase in reagent volume cause a slight decline in the RFI. This implies that the optimal value to be used is one ml of 0.02%w/v fluorescamine. (Figure 5)

3.1.4. Time of reaction’s effect In this study, the formed fluorophore’s formation and stability were affected by the period of time (0 to 25 minutes) allowed for the reaction to proceed. The reaction showed the greatest fluorescence at 8 minutes and remained steady till 20 minutes, beyond which a slight decline in RFI values occurred. (Figure 6)

3.1.5. Diluting solvent’s effect Various solvents were used as the diluent for the final reaction product to discover the most appropriate one that achieves the greatest RFI values. Among the diluents tried were distilled water, ethanol, methanol, acetone, acetonitrile, and DMF. From the obtained results, ethanol was considered the preferable diluent of choice as it showed the highest RFI values.

3.2. Determination of the reaction’s stoichiometry Utilizing equimolar concentrations (4.0 X 10-4 M) of both the drug and reagent, Job’s method was applied to construct a plot between the reaction product’s fluorescence intensity versus the mole fraction of heptaminol (figure 7). From this plot, it was detected that the maximum fluorescence intensity value appeared at the mole fraction 0.5 implying that fluorescamine and heptaminol react at a ratio 1:1 as presumed in the suggested pathway. (Figure3)

3.3. Validating the proposed method The optimized proposed method was validated following ICH guidelines [27] via determining various parameters that include :

3.3.1. Linearity and range After using all the previously determined optimized conditions, a standard calibration curve was constructed using the RFI values measured at 471 nm (after excitation at 393 nm) versus their corresponding concentrations of heptaminol standard solutions. This plot was linear over the concentrations ranging from 75 to 850 ng/ml (r2 = 0.9994). Table 1 listed the other analytical parameters that were determined.

3.3.2. Accuracy The presented method was done on 6 concentration levels (100, 150, 250, 350, 500, 600 ng/ml) within the linear range. The mean recovery percentage of three replicates performed for each concentration level was calculated. Higher values of the recovery percentages calculated along with the decreased standard deviation values indicate the high accuracy of the proposed approach as listed in table 2.

3.3.3. Precision The proposed method was repeated 3 times during the same day and 3 times in 3 successive days on 5 concentration levels (150, 250, 350, 500, 600 ng/ml) present within the method’s linearity to determine its precision. Whether inter-day or intra-day results, they both showed a relatively high recovery percentage with percentage relative standard deviations lower than 2 % as

mentioned in table 3. This indicates the repeatability and reproducibility of the proposed approach and its reliability for quality control analysis.

3.3.4. Sensitivity The sensitivity of the presented approach was determined via calculating the limits of detection and quantitation (LOD and LOQ), following ICH guidelines[27], through the application of the underneath equations: *LOD=3.3 X ơ/S *LOQ=10 X ơ/S Where S is the slope of the constructed calibration curve and ơ is the standard deviation of its intercept. For the proposed method the calculated values of LOD, LOQ were 23.85 and 72.29 ng/ml respectively. This significantly implies that the presented approach is highly sensitive.

3.3.5. Robustness Deliberate slight changes were done in various experimental factors without affecting the RFI values measured by the presented method indicating its robustness. Among those experimental factors were pH (± 0.2 pH units), fluorogenic reagent volume (± 0.2 ml) and the time allowed to get a complete reaction (± 2 minutes). This was elucidated in table 4 in %recoveries that showed %RSD lower than 2%, thus indicating the proposed method to be robust.

3.4. Applications of the presented method 3.4.1. Applications to analyze heptaminol in its tablets compared to a reported method. Corasore® (150 mg) tablets analyzed using the proposed approach was showing a high mean percentage recovery of 99.47% ± 1.25. When compared to a reference reported method[8] with respect to t and f values at 95% confidence interval, the statistically calculated t and f values were less than their tabulated values (2.78 and 6.39 respectively).Thus, implying the good accuracy and precision of the presented method. (Table 5)

3.4.2. Application to spiked plasma

Owing to the higher sensitivity of the presented method, it was utilized to determine heptaminol in spiked human plasma on 3 concentration levels present within its linear range. The concentrations of the cited drug were estimated via the application in the regression equation. For each concentration, the percentage recoveries were calculated. The mean value of the calculated percentage recoveries was 100.52 ± 1.19%, thus indicating the possibility of determining heptaminol in human plasma using the presented method. (Table 6)

4. Conclusion The presented study provided a relatively economic time efficient method for the analysis of heptaminol, in its pure form or in its tablets or in human plasma spiked with the cited drug, where there is neither the need for heating nor further extraction. The presented method also uses less expensive instruments, solvents and reagents when compared to other reported methods. Its high accuracy, precision, sensitivity, and feasibility make it a reliable and reproducible method to be used in quality control analysis of heptaminol.

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[10] S.M. El-Adl, A new sensitive fluorimetric method for the determination of heptaminol and mexiletine in pharmaceuticals, Scientia Pharmaceutica 70 (2002) 57-65. [11] X.H. Yang, X.C. Wang, X.M. Zhang, Capillary zone electrophoresis separation of low concentration stimulants in human urine with laser-induced fluorescence detection, Analytica Chimica Acta 549 (2005) 81-87. https://doi.org/10.1016/j.aca.2005.06.008 [12] O.M. El-Houssini, N.H. Zawilla, M.A. Mohammad, Development and Validation of RP-LC Method for the Determination of Cinnarizine/Piracetam and Cinnarizine/Heptaminol Acefyllinate in Presence of Cinnarizine Reported Degradation Products, Analytical Chemistry Insights 8 (2013) 99-106. https://doi.org/10.4137/aci.s12478 [13] K.F. Hsu, K.Y. Chien, G.P. Chang-Chien, S.F. Lin, P.H. Hsu, M.C. Hsu, Liquid chromatography-tandem mass spectrometry screening method for the simultaneous detection of stimulants and diuretics in urine, Journal of Analytical Toxicology 35 (2011) 665-674. https://doi.org/10.1093/anatox/35.9.665 [14] Y.L. Tseng, C.H. Liu, F.H. Kuo, M.H. Shieh, Solid-phase column chromatographic and gas chromatographic-mass spectrometric determination of heptaminol in human urine and related pharmacokinetic profiles, Journal of Analytical Toxicology 30 (2006) 365-369. https://doi.org/10.1093/jat/30.6.365 [15] S. RabouanGuyon, P. Courtois, D. Barthes, Determination of acefylline heptaminol in pharmaceutical preparations by high performance liquid chromatography, Farmaco 51 (1996) 739-746. [16] A. Morros, L. Borja, J. Segura, Determination of heptaminol in plasma by thin-layer chromatography and in situ fluorimetry, Journal of Pharmaceutical and Biomedical Analysis 3 (1985) 149-156. https://doi.org/10.1016/0731-7085(85)80018-2 [17] M. Cociglio, D. Sauvaire, R. Alric, Liquid chromatographic assay of heptaminol in serum and its oral pharmacokinetics in the dog, Journal of Chromatography B: Biomedical Sciences and Applications 307 (1984) 351-359. https://doi.org/10.1016/S0378-4347(00)84106-7 [18] P. Leroy, A. Nicolas, A. Moreau, electrochemical detection of sympatomimetic drugs, following precolumn o-phthalaldehyde derivatization and reversed-phase high-performance liquid chromatography, Journal of Chromatography A 282 (1983) 561-568. https://doi.org/10.1016/S0021-9673(00)91632-7 [19] R.R. Brodie, L.F. Chasseaud, L. Rooney, A. Darragh, R.F. Lambe, Determination of heptaminol in human plasma and urine by high-performance liquid chromatography, Journal of Chromatography B: Biomedical Sciences and Applications 274 (1983) 179-186. https://doi.org/10.1016/S03784347(00)84421-7 [20] A. Nicolas, P. Leroy, A. Moreau, M. Mirjolet, Determination of heptaminol in pharmaceutical preparations by high-performance liquid chromatography, Journal of Chromatography A 244 (1982) 148152. https://doi.org/10.1016/S0021-9673(00)80132-6 [21] J. Rabiant, M. Sergant, A.F. Gaudin, Heptaminol determination by gas chromatography: application to the study of urinary excretion, Annales Pharmaceutiques Francaises 29 (1971) 331-336. [22] M.A. Omar, D.M. Nagy, M.E. Halim, The convenient use of fluorescamine for spectrofluorimetric determination of midodrine hydrochloride in pure form and its tablets formulation: Application to content uniformity testing. , Luminescence,1522-7243 (Electronic) (2018). https://doi.org/10.1002/bio.3582 [23] F.F. Mohammed, Khalid M. Badr El-Din, S.M. Derayea, Switch on fluorescence probe for the selective determination of lisinopril in pharmaceutical formulations: application to content uniformity testing, RSC Advances 8 (2018) 16269-16277. https://doi.org/10.1039/C8RA01617H [24] A.G. Palekar, A fluorometric method for measurement of dipeptidase activity, Analytical Biochemistry 104 (1980) 200-204. https://doi.org/10.1016/0003-2697(80)90299-7 [25] R.F. Chen, Smith, Paul D.,Maly, Mark, The fluorescence of fluorescamine-amino acids, Archives of Biochemistry and Biophysics 189 (1978) 241-250. https://doi.org/https://doi.org/10.1016/00039861(78)90209-6

[26] S. Udenfriend, S. Stein, P. Böhlen, W. Dairman, W. Leimgruber, M. Weigele, Fluorescamine: A Reagent for Assay of Amino Acids, Peptides, Proteins, and Primary Amines in the Picomole Range, Science 178 (1972) 871. https://doi.org/10.1126/science.178.4063.871 [27] ICH harmonized tripartite guideline: Guideline for Good Clinical Practice.

Table 1. Analytical parameters obtained from the proposed fluorimetric method. Parameters

Proposed method

Excitation wavelength (nm)

393

Emission wavelength (nm)

471

Linear range (ng/ml)

75-850

Correlation coefficient (r)

0.9994

Determination coefficient (r2)

0.9988

Slope± SD

0.78 ± 0.01

Intercept ± SD

35.52 ± 5.69

LOD (ng/ml)

23.85

LOQ (ng/ml

72.29

Table 2. Evaluating the accuracy of the proposed method at 6 concentration levels within the linear range. %recoverya ± SD

Sample number

Concentration level (ng/ml)

1

100

100.75 ± 1.41

2

150

100.16 ± 1.40

3

250

100.55 ± 1.35

4

350

99.88 ± 1.02

5

500

99.96 ± 1.34

6

600

100.28 ± 0.48

a

Mean of six determinations.

Table 3.Intra- and inter-day precisions for the proposed method. Concentration

Intraday precision

(ng/ml)

%recoverya

RSD

%recoverya

RSD

150

100.52

1.14

98.88

1.40

250

99.89

0.90

100.17

0.77

350

100.12

1.11

102.20

0.84

500

99.89

1.56

99.00

1.05

600

100.05

0.40

100.74

1.06

a

Mean of four determinations.

RSD = relative standard deviation.

Interday precision

Table 4. Robustness for determination of Heptaminol using the proposed method Method parameters

%recovery a ± RSD

Buffer pH 8.8

99.45 ± 0.55

9.0

100.30 ± 0.04

9.3

99.81 ± 0.95

Volume of fluorescamine (ml) 0.8

99.79 ± 0.01

1.0

100.00 ± 0.29

1.2

99.72 ± 0.14

Time (minutes)

a

8

99.69 ± 0.77

10

99.21 ± 1.64

15

99.55 ± 1.05

Mean of three determinations.

Table 5. Proposed and reported methods used for the determination of heptaminol spectrofluorimetrically. Mean %recovery ± SDa Dosage form

Corasore ®

Labelled

Proposed

Reported

content

method

method

150 mg

99.47 ± 1.25

98.16 ± 1.17

t-valueb

f-valueb

1.68

1.14

tablets a

Mean of five determinations.

b

The tabulated t‐ and f‐values at the 95% confidence limit are 2.78 and 6.39, respectively.

Table 6. Application of the proposed method for the determination of heptaminol in spiked human plasma Concentrations added (ng/ml)

a

% Recovery a ± SD

250

101.28 ± 1.95

500

101.39 ± 1.95

750

99.96 ± 0.57

Mean ± SD

100.52 ± 1.19

%RSD

1.18

Mean of three determinations.

H HO

NH2

Figure 1. Chemical structure of heptaminol.

800 Drug emmision Drug excitation Blank emmision Blank excitation

RFI

600

400

200

0 300

350

400

450

500

550

600

Wavelength (nm)

Figure 2. Excitation (393 nm) and emission (471 nm) spectra of the products of the reaction between heptaminol (750 ng/ml) and fluorescamine.

OH

O O O H HO

NH2

O

Fluorescamine reagent

H NH O

Heptaminol N

O COOH

fluorescent product

Figure 3. Suggested pathway of the reaction between heptaminol and fluorescamine.

Volume of buffer 0

0.3

0.5

0.7

1

1.2

1.5

2

2.5

250

RFI

200

150

pH buffer vol

100

50

7.7

8.2

8.6

8.8

9

9.3

9.5

9.7

10

10.2 10.7

12

pH Figure 4. Effect of PH and buffer volume on the RFI of the product of the reaction between heptaminol (250 ng/ml) and fluorescamine using borate buffer.

240

220

200

RFI

180

160

140

120

100 0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Volume of fluorescamine

Figure 5. Effect of volume of 0.02%W/V fluorescamine on the RFI of the reaction product of heptaminol (250 ng/ml).

1.8

240 220

RFI

200 180 160 140 120 100 0

5

10

15

20

25

Time (min)

Figure 6. Effect of time on the RFI of the product of the reaction between heptaminol (250 ng/ml) and fluorescamine.

30

350

300

250

200

150

100

50

0 0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Figure 7. Job’s method for the determination of the stoichiometry of the reaction between heptaminol and fluorescamine with equimolar concentrations of 4.0 X 10-4 M.

(1) Development of sensitive and simple spectrofluorometric method for determination of heptaminol (2) The proposed method based on the reaction between fluorescamine reagent with the primary amine group found in heptaminol (3) The proposed method was validated of according to the ICH guidelines. (4) the presented approach was adapted for in vitro study of heptaminol in spiked human plasma

The Presented work represent one of important, simple, time saving and innovative method for quality control analysis of heptaminol.