Continuous Flow Injection Analysis Method for the Determination of a Drug Diphenhydramine Hydrochloride by Using Phosphomolybdic Acid

The study presented a new method for determining diphenhydramine hydrochloride (DPH) in its pure form and some pharmaceutical preparations, within CFIA technique. The method is simple, fast, sensitive, easy to operate, and of a low cost. It is based on the reaction of DPH with phosphomolybdic acid (PMA), in aqueous media, forming a white, slightly yellowish precipitate. The precipitate formed was studied using an Ayah 6S×1-ST-2D solar cell CFI analyzer, by the reflection of incident light off the surfaces of the deposited particles at (0–180°), expressed as the response measured in (mV) . Some chemical and physical variables were studied to provide optimal conditions for the study. The linear range was 0.07-9 mmol/L and it had a correlation coefficient (r) with a value of (0.9998). The limit of detection (L.O.D.) of the new method was 729.55 ng/sample, calculated by gradually diluting the lower limit of the concentration in the linear range (0.07 mmol/L). The % RSD was less than 0.2% for a concentration of 0.1, 4.0 and 10.0 mmol/L of DPH for n = 8. The method was successfully applied in the determination of DPH in three samples from three different pharmaceutical production companies. Using the standard addition method, the new method was compared with the UV-Vis spectrophotometry method at λmax = 258 nm. Both T-test and F-test. The results of the two tests showed no significant difference at a confidence level (95%).


Introduction
Diphenhydramine hydrochloride (DPH), which is known by the chemical name 2-(Diphenylmethoxy)N,N-dimethylethanamine hydrochloride molecular structure as in Fig. 1 , its chemical formula is C17H22ClNO , the general appearance of it in its pure form is a white crystalline powder .Highly soluble in water, it's freely soluble in alcohol.It's offered commercially as a pharmaceutical preparation in several forms, including the form of tablets or syrup taken orally , or in the form of injections taken through intramuscular or intravenous injection 1,2 .It's one of the antihistamines and falls into the category of the first generation of antihistamines 2,3 .It's used alone or in combination with other medicine 4 .Diphenhydramine hydrochloride is prescribed to relieve allergy symptoms , to treat motion sickness, as an anti-emetic and anti-couch ,in cases of bee stings and skin itching and to treat some cases of runny nose and eyes 5 .There are several methods for estimating diphenhydramine hydrochloride in literature; we mention some of them: spectrophotometric methods 6,7 , chromatographic methods [8][9][10][11] , flow injection 12, 13 and potentiometric methods 14 .In this study, a new method was introduced within the continuous flow injection analysis technique [15][16][17][18] ,using homemade cells [19][20][21][22] , which depends on fluorescence or turbidity [23][24][25][26] as it is an easy, simple, sensitive, safe and inexpensive method to control the quality of DPH-containing drug tablets using homemade Ayah 6S×1-ST-2D solar cell CFI analyzer 27 , which has been successfully used in the determination of some drugs and some elements 28 .The study aims to present a simple, easy-to-work, environmentfriendly, low-cost, repeatable and high recovery percentage analytical method for diphenhydramine hydrochloride (DPH).

Sample Preparation
A quantity of twenty tablets was weighed with a four-stage sensitive scale, crushed, ground using a mortar, and used a 200 mesh sieve to sieving the formed.Each tablet has 25 mg of diphenhydramine hydrochloride (supplier from SDI -Iraq, Aswar Al-Khaleej-Iraq and Al-Kindi-Iraq) weight :0.6659, 0.6875, & 0.6968 g according to the sequence, which is equivalent to 0.14591 g of the active drug substance to get 10 mmol/L.Distilled water was used to dissolve the powder and to get rid of any undissolved residue that might affect transducer response, the solution was filtered.These solutions were prepared in a 50 ml volumetric flask (The volume was completed to the mark in a 50 ml volumetric flask with distilled water before the filtration process).

Apparatus
The apparatus used in the new method for DPH determination consists of a two-channel variable speed peristaltic pump (supplied from Ismatec -Switzerland) and three pairs-hole medium pressure injection valve (IDEX Co.-U.S.A. supplied it) with a sample loop (0.7 mm i.d.Teflon, different length).The response was measured using Ayah 6S×1-ST-2D Solar cell CFI analyser (homemade), containing six snow white LEDs as a light source in a 2mm flow cell.Two solar cells were used as a detector to collect signals via a 60 mm travel-sample.The response is output in the form of peaks through the x-t potentiometric recorder (Kompenso GraphC-1032, supplied from Siemens-Germany, 1-500 volt,1-500 mV) or Digital AVO

Methodology
Two lines make up the manifold flow system Fig. 2 for determination of DPH by formation ion-pair complex with Phosphomolybdic acid (PMA) in the aqueous medium, where a white slightly yellowish precipitate was formed.The first line supplied distilled water, which is connected to the injection valve as a carrier stream moving at 2.0 ml/min for hold DPH (used sample volume 50 µl), the second line carried PMA (20 mmol/L) at 2.0 ml/min .At Y-Junction , the reagent line and the sample current line meet, where the starting point of the reaction is, after which the reaction product heads towards the measurement cell in Ayah 6S×1-ST-2D Solar cell CFI analyser, each solution was injected three times succession .The responses were captured by x-t potentiometric recorder and the responses appeared in the form peaks whose height is proportional to the amount of light reflected from the surfaces of the precipitate particles after the light fell on them from the source.A mechanism has been suggested for the reaction between DPH and PMA in aqueous medium as shown in scheme 1 14 .
Scheme 1.The suggested mechanism of the DPH and PMA reaction in aqueous media medium

Results and Discussion
The concentration of the PMA reagent and the selection of the type of reaction medium (carrier stream), were mainly studied to determine some of the optimal chemical parameters.While the flow rate, sample volume and purge time, were studied to https://doi.org/10.21123/bsj.2024.8926P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal determine some of the optimal physical parameters.By making it constant and variable each time these variables were optimized.

Effect of Phosphomolybdic Acid (PMA) Concentration
A range of PMA concentrations between 1 and 25 mmol/L was generated to determine the ideal reagent concentration.Distilled water was employed as a carrier stream with a sample volume of 25 µl.A (6 mmol/L) DPH concentration was injected, both the carrier and reagent lines at a flow rate of 2.3 ml/min.Three times of each measurement were done.

Effect of Different Medium
In order to determine the optimal medium for the interaction between diphenhydramine hydrochloride (6 mmol/L) with phosphomolybdic acid (20 mmol/L) , this experiment was carried out using solutions of different acids and salts as a carrier stream, which were (CH3COOH , HCl , HNO3 , H2SO4 , NaCl ,NH4Cl ,NaNO2 ,NaNO3 ,Na2CO3) at a concentration of 0.1mol/L, as well as aqueous medium .Observed through the results of the experiment ( shown in plot of the transducer response against time) in Fig. 4A.Different media except for the aqueous medium caused a decrease in the S/N response; This can be explained by the fact that these media caused more agglomeration of the sediment particles and fusion with each other, as part of the reflector is lost and a decrease in the amount of light reflected on the surfaces of the sediment particles and thus a decrease in the transducer response (decrease in peak height), while the distilled water caused the sediment particles to not agglomerate and to increase the regularity of their shape, and the amount of light reflected from their surfaces is greater.Thus, the higher response appeared when distilled water was used as a reaction medium (carrier stream).Therefore, subsequent experiments used distilled water as the optimal carrier stream.A summary of the experiment results is shown in Table 2.

Flow Rate
Flow rate is one of the physical variables affecting the interaction of DPH determination.After examining the effects of flow rate on the transducer's response, the optimal flow rate for this experiment will be selected.The experiment was within the extent (1.0-2.8 ml/min) for both the reagent and carrier streamlines.A peristaltic pump controls the flow rate, the concentration of DPH was 6 mmol/L, 25µl of the sample was utilized in this experiment and the reagent (PMA) was used at 16 mmol/L.The carrier stream was distilled water.Fig. 5A shows how varying the flow rate affects the transducer's response.The experiment's results are summarized in Table 3, which shows that the flow rate 1.5 ml/min for each of the two lines produced the highest response. .Therefore, it was adopted as an optimal flow rate in subsequent experiments.

Sample Volume
In this experiment, the concentration of DPH (6 mmol/L) , the concentration of PMA (16 mmol/L) and distilled water were used as a carrier stream to test the effect of changing the sample volume on the intensity of the response, using the optimal parameters that were established in the previous experiments , such as (1.5 ml/min ) the optimal flow rate for both the PMA line and the carrier stream line .It's different for the sample loop in the injection valve , it can be observed in the graph of the response against time Fig. 6A that as volume increases, the peak was sharp (needle peak), when the sample volume (50µl) , as explained in Fig. 6A and through Fig. 6B it was observed when using a sample volume greater than (50µl) , there is a relatively constant rise in the response and breadth of the base , so the volume of (50µl) will be adopted as an optimal sample volume in subsequent experiments .A synopsis of the experiment's results shown in Table 4.

A Calibration Curve (Scatter Plot) For the Variance of DPH Concentration against Energy Transducer Response
The optimal chemical with physical parameters were adopted to prepare a series of DPH solutions within the extent (0.07-10) mmol/L , the measurement was repeated for each concentration three consecutive times .Responses are shown as in Fig. 8A, which shows the response range and peak height of each concentration of DPH.As shown in Fig. 8B, the linear calibration curve was within the range (0.07-9)mmol/L, accompanied by a correlation coefficient (r)=0.9998.The obtained results ( which briefly show the linear regression of the change of transducer response against the change of DPH concentration , the equation that was used in this part of the study was y ̂=a+bx 29 (first-degree equation).The value of t was calculated at a confidence level (95%), which was greater than the value of tabular t, which leads us to say that linearity versus nonlinearity is acceptable.Synopsis of results is shown in Table 6.

Limit of Detection (L.O.D)
This study was carried out to determine the detection limit for DPH using three different approaches.: practically based on gradual dilution of the minimum concentration in the calibration curve, it was calculated theoretically on the basis of the value of the slope and on the basis of the linear equation, and the results of this part of the work are summarized in Table 7.The minimum concentration was 0.07 mmol/L and the sample volume was 50µl.

Repeatability
The percentage equivalent to the test-retest measurement reliability expresses the relative standard deviation.The responses were re-measured for each concentration through eight consecutive injections for three stable DPH concentrations (0.1, 4.0 and 10.0) mmol/L in optimum conditions for n=8.Response outline shown in Fig. 10, Table .8, indicates that the relative standard deviation as a percentage was less than 0.2%, making it abundantly evident that the suggested approach and instrument were suitable for determining DPH.

UV-Spectrophotometric
In order to evaluate the new DPH determination method, a comparison was made between it and a UV-spectrophotometric method, on which absorbance measurements are based.The concentration extent of the method was 0.05-0.9mmol/L at λmax=258 nm at 0.55 mmol/L, Fig. 10, using quartz cell(1ml) .From Fig. 11 , the scatter plot from 0.05-0.9mmol/L, while the linear extent was (0.05-0.65 mmol/L) .Correlation coefficient (r) = 0.9994 and R 2 %= 99.90 , n=14 (n= number of measurements) .The detection limit was 8.7546 µg/sample; it was calculated by gradually diluting the minimum concentration in the calibration curve (0.05 mmol/L).Synopsis of the results of the method is illustrated in Table .9To determine DPH in pharmaceutical preparations, to evaluate the efficiency of the new method, which was made using Ayah 6S×1-ST-2D Solar cell CFI Analyser(homemade), five solutions were prepared for each drug for samples from three different companies for the production of pharmaceutical preparations (SDI-Iraq , Aswar Al-Khaleej-Iraq and Al-Kindi-Iraq) , where comparison was made with the UV-Spectrophotometric method (classical method) after applying the method of a standard addition to both the two methods are as follows: For the new method: five volumetric flasks of 10 ml were prepared and 3.0 ml from 10 mmol/L was transferred into each of them, after which different volumes of the standard solution of DPH were gradually added (S.D.I.-Iraq) (0 , 2 , 3 , 4 , 5 )ml of 10 mmol/L to gain 0, 2 , 3 , 4 , 5 mmol/L.
For the classical method: five volumetric flasks of 10 ml were prepared and 1 ml from 1 mmol/L was transferred into each of them, after which different volumes of standard solution of DPH were gradually added (S.D.I.-Iraq) (0, 1, 2, 3, 4 )ml from 1 mmol/L to gain 0 , 0.1 , 0.2 , 0.3 , 0.4 mmol/L.
Flask number 1 is sample.Measurements were made for both methods.The results obtained from the standard addition method were mathematically processed.The results are summarized in Table 10A, B at C.I. (95%) The t-test and F-test were used to evaluate whether there is a significant difference.The results were processed statistically 29 .The results of the t-test and F-test were summarized in Table 10B (columns 4 and 5).The findings made it very evident that, at a 95% level of confidence, there is no significant difference between the new method and the classical method, t-calculated (1.846) less than t-tabular (4.303), as well as the calculated F-value (1.1682) less than tabular Fvalue (39).Fig. 12 shows that, for three drug samples, the effect of changing DPH concentration on the S/N energy transducer response against time using the Ayah 6SX1-ST-2D solar cell CFI analyser.---------------

Conclusion
The method described in this study is simple, sensitive, speedy, requires a few samples and regents, and is simple to use.When distilled water was used as the reaction medium, it was successfully applied to the measurement of diphenhydramine hydrochloride in both pure and pharmaceutical preparations based on the formation of a white-slightly yellowish precipitate as a result of the interaction of DPH with phosphomolybdic acid (PMA).The findings of the study demonstrated that the new method is a viable substitute for the classical method with which it was compared, i.e., an alternative analytical method was discovered through this study, which was carried out under simple conditions and with optimum parameters using an Ayah 6SX1-ST-2D solar cell CFI analyzer.

Figure 2 .
Figure 2. Flow diagram manifold system used for determination of diphenhydramine hydrochloride(DPH) Fig. 3A` depicts the experiment's response diagram.Table.1 provides a summary of the findings.The plot of results using the Ayah 6S×1-ST-2D solar cell -CFI Analyser is shown in Fig.3A.The responses outlined in Fig. 3A demonstrated that the response peak heights gradually increased until the concentration (20 mmol/L) at which the response peaked, where it showed a peak with the highest height and lowest base width of the other responses.As a result, the concentration of the reagent 20 mmol/L was determined to be the best in this experiment and will be utilized in further tests.Fig. 3B revealed the effect of PMA concentration on the transducer response's peak rising rate.PMA concentration's effect on: A: Response outline against time.B: Peak rise rate of transducer response in (mV).Table 1.Synopsis of the results of effect of the concentration of PMA on the responses average of the transducer.: January, 2024 https://doi.org/10.21123/bsj.2024.8926P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal PMA: Phosphomolybdic acid . ̅ i(mV)(S/N) response of an energy transducer in (mV) for n=3,ttab0.05/2,2=4.303.RSD%:relative standard deviation, σn-1:standard deviation, n: the number of repetitions of the measurement, Interval of confidence: A range of values above and below the point estimate where the true value is most likely to be found with a 95% confidence level Fig. 4B reveals the effect of different media on the rate of increase of the transducer response peak.type's effect on: Response outline against time.B:A graphical diagram showing the effect of medium change on the response average of a transducerTable 2. Synopsis of the results of the effect of changing the mediums on the response of the energy ̅ i(mV)(S/N)response of an energy transducer in (mV) for n = 3, ttab0.05/2,2= 4.303.RSD%:relative standard deviation, σn-1:standard deviation, n:the number of repetitions of the measurement, Interval of confidence: A range of values above and below the point estimate where the true value is most likely to be found with a 95% confidence level Physical Variables Fig. 5B emphasizes the effect of flow rate on the transducer response's peak rising rate, base breadth ∆tB and arrival time to the measuring flow cell.Figure 5. Flow rate's effect on: A: response outline against time.B:peak rise rate of transducer response in (mV), base breadth ∆tB(sec) and arrival time to the measuring flow cell (sec) Table 3. Synopsis of the results of the effect of changing the flow rate on the response of the transducer.
̅ i(mV) (S/N) response of an energy transducer in (mV) for n=3,ttab0.05/2,2=4.303.RSD%:relative standard deviation, σn-1:standard deviation, n: the number of repetitions of the measurement, Interval of confidence: A range of values above and below the point estimate where the true value is most likely to be found with a 95% confidence level, t: Time arrival estimated from the injection valve to the measurement cell (sec),∆t: Base breadth of peak(sec), Vadd: Addition volume(ml)in flow cell, Df: Dilution factor in flow cell Fig. 6B indicated that the sample volume affected the transducer response's peak increase rate.and arrival time to the measuring flow cell.(A) https://doi.org/10.21123/bsj.2024.8926P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal (B) Figure 6.Sample volume's effect on: A:Outline's response against time .B: Peak rise rate of transducer response in (mV) , base breadth ∆tB (sec) and arrival time to the measuring flow cell (sec).Table 4 .Synopsis of the effect of sample volume change on transducer response average results repetitions of the measurement, Interval of confidence: A range of values above and below the point estimate where the true value is most likely to be found with a 95% confidence level, t: Time arrival estimated from the injection valve to the measurement cell (sec), ∆t: Base breadth of peak(sec), Vadd: Addition volume(ml)in flow cell, Df: Dilution factor in flow cell Purge Time In this experiment, different times were used to purge the sample section (0.45-8 sec) and the opening position of the injection valve (10 sec).The carrier solution passes through the injection valve https://doi.org/10.21123/bsj.2024.8926P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal when the valve is in the injection position, followed by turning the valve to the loading position.The experiment was carried out using the optimal parameters established in previous experiments.The concentration of DPH used in this experiment was 6 mmol/L.A synopsis of the results of the experiment is shown in Table 5, Fig. 7A shows the transducer's response against time, which shows a continuous increase in response with increased purge time up to the time of 10 sec which is the open valve mode.Therefore, it was chosen as the optimal time to purify the sample from the sample loop completely.Fig. 7B shows the effect of purge time on the peak rise rate of transducer response, base breadth ∆tB and arrival time to the measuring flow cell.purge time's effect on: A:Response outline against time.B:peak rise rate of transducer response in (mV), base breadth ∆tB(sec) and arrival time to the measuring flow cell (sec) Table 5. Synopsis of the results of the effect of changing the purge time on the response of the transducer A: A calibration curve shows the effect of DPH concentration change against time on transducer response (for some responses) (B) Figure 8. B: Scatter and linear plot of the new method expresses the transducer response against concentration by linear equation .
µg/50µl X= limit of detection value , SB=standard deviation value of blank refined for 16 times, Yb: average response for blank=intercept(a), Sb: Standard deviation equal to Sy/x(residual) from linear range , ̂:estimated response(mV).

range of values above and below the point estimate where the true value is most likely to be found with a 95% confidence level, t: Time arrival estimated from the injection valve to the measurement cell
̅ i(mV) (S/N) response of an energy transducer in (mV) for n=3,ttab0.05/2,2=4.303.RSD%:relative standard deviation, σn-1:standard deviation, n: the number of repetitions of the measurement, Interval of confidence: A (sec), ∆t: Base breadth of peak(sec) https://doi.org/10.21123/bsj.2024.8926P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal