Ciprofloxacin assessment via the formation of precipitate reaction product

with ammonium metavanadate as a reagent using ISNAG-Fluorimeter

Abstract The assessment of Ciprofloxacin.HCI By using continuous flow injection

diverging light. A moder, simple, rapid, and sensitive approach has been developed by
using an ISNAG-Fluorimeter analyzer for assessing Ciprofloxacin.HCI pure state as well
as pharmaceuticals (tablets) , the process was based on the interaction of
Ciprofloxacin.HCI with ammonium metavanadate (v) (AMV) as a precipitating agent to
yield an ion pair association yellow precipitate rapidly in salt medium. To increase the
affectability of the established approach, optimum parameters have been selected. For the
instrument response vs Ciprofloxacin-HCl concentrations, the scatter plot range and linear
dynamic range were 0.0- 50 mmol/L. For linear dynamic range, the correlation coefficient
(r) was 0.98623, and the percentage linearity (R 2 percent) was 97.26%. With a
concentration of 10, 35 mmol/L, the percentage linearity for repeatability (n=8) was less
than 0.34 percent. The detection limit of gradual dilution for the lowest concentration in the
scatter plot range of the calibration curve was 0.221g/sample. Ciprofloxacin.HCI in
pharmaceutical tablets was effectively estimated using the described approach. The
individual t-test with 95 percent confidence (α=0.05) was used to analyze drugs using the
usual incremental technique . The results were also compared to those obtained using
traditional UV-Spectrophotometric techniques at a wavelength of 272 nm. The ISNAG-
Fluorimeter analyser and traditional techniques yielded no significant differences in the
results.

Keywords: Ciprofloxacin.HCI, flow injection, diverge light, ISNAG-Fluorimeter

analyzer.

1. Introduction
Ciprofloxacin hydrochloride , a fluoroquinolone, a broad spectrum second generation
microbiological agent activity against Gram-positive and Gram-negative bacteria such as
Pseudomonas aeruginosa, Streptococcus faecalis, Staphylococcal aureus, and Enterobacter
aerogenes. Antibiotic Ciprofloxacin complex exerts its disinfectant effect by inhibiting the
synthesis of DNA in bacteria, preventing their growth and preventing the spread of infection.
tablets have been marketed since 1987 for the treatment of a wide variety of infectious diseases
in adults.[1]
IUPAC Name: 1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-
quinolinecarboxylic acid [1]. Molecular Formula: C 17H19ClFN3O3[2].Chemical and physical
properties: Trade names: Ciloxan, Cipro, Neofloxin, others, State: Solid (i.e., white powder
with bitter taste.), Molecular Weight: 367.802 g/mol, solubility: soluble in acetic acid, and
slightly soluble in water, methanol, ethanol, or acetone, Melting point (°C): 255-257pKa
(Strongly Acidic)5.76 , pKa (Most grounded Basic) 8.68 and biological half-life 3.5 h [3,4]
Utilization:-urinary tract infections, ophthalmic, respiratory, bone and joint , intraabdominal
infections, bacterial diarrheal diseases and periodontal microorganisms [5,6] Side effect: CNS
Nervousness, agitation, anxiety, confusion, depression, orthostatic hypotension, vasculitis,
Blurred vision, burning, stinging, irritation, nausea, vomiting, diarrhea, constipation,
1
discomfort, albuminuria, candiduria, renal calculi, rashes, exfoliative dermatitis, erythema . [5,
7] .
The aim of the present study: establish a new, accurate , sensitive and simple method by Flow
injection analysis combine with ISNAG-Fluorimeter analyzer for determination Ciprofloxacin.HCl as
Ionic pair precipitate by using metavanadate (v) (AMV) as a reagent.

Materials and methods: Analytical reagents and all chemical substances were used, and
distilled water was used in all solutions., C17H18FN3O3.HCl. 3.8580 g /100 mL (SDI) , Na2CO3
5.299g /100ml (Fluka), CH3COONH4 3.884g /100M (BDH), KCl 7.350 g/100mL
(Hopkin&Williams),NaCl 7.313 g/250mL(BDH), NH4Cl 2.675g/100 mL (BDH),, CH3COOH
57.47 ml/L (BDH),, HCl 88.28 ml/L (BDH), Tartaric acid (C4H6O6) 7.513mg/200ml (T.Homas
baker), ascorbic acid (C6H8O6) 8.807gm/200ml (Himedia) and H2SO4 55.52 ml/L (BDH),.

Sample preparation of Ciprofloxacin. hydrochloride (CIP.HCl)

Twenty tablets, containing 500 and 750 mg CIP.HCI, were weighted, then crushed and ground,
resulting in 0.9195 g of active ingredient (50 mmol/L) in each batch for ( AL-jazeera-750mg-Iraq ,
Micro labs limited -500mg- India, citro pharma Inc-500mg- Canada and pioneer-500mg -Iraq). A
solution of distilled water was made to dissolve the powder. The volume was increased to 25 mL in
order to remove any undissolved materials that may have affected the calibration. A hot distilled water
wash was performed on the residue, and volume was increased to 50 mL with the same solvent (distilled
water).

Apparatus:
 Two-line sophisticated design system for precipitating agent and CIP use. The nuances
of the complex used were depicted in HCI figure1.
 Two speed-changing channels in the peristaltic pump (Ismatec, Switzerland).
 6-port medium-pressure injection valve with test loop (I D E X corporation, USA)
(1mm i.d. Teflon variable length)
 The yield response was recorded using a potentiometric recorder (Siemens, Germany).
 UV light spectrophotometer (Shimadzu 1800).
Methodology: ciprofloxacin.HCl was tested in a two-line manifold unit configuration
using ammonium metavanadate (v) (AMV) as a precipitating agent. Yellow precipitate
is most likely formed as an ion pair (Figuer1). A carrier stream will pass through the
injection valve to drain the sample segment (150 mL with open valve mode) with 12
mmol/L CIP.HCl concentration and connect with a second stream at the Y- junction
point to create the precipitate (3.20 ml flow rate for each line). This precipitate will be
exposed to two primary wavelengths, 184.9nm and 253.7nm (prominent). Because of
their high frequency, both lines are readily diverged. All types of dispersed light will be
detected at 90o using a route of 2mm optical aperture stretched for 100 mm distance
using two sides solar cells, each side containing four solar cells (i.e., 2[4x2.5cm (long)
[8-4]. Sketch 1 depicts the mechanism of reaction.

Figuer.1: The flow gram of the manifold was employed throughout this investigation.

2
Scheme 1: Proposal mechanism for the reaction between Ciprofloxacin.HCl and ammonium
metavanadate (V) [15,16]
Rustle and discussion
Chemical variables
Effect of ammonium metavanadate (V) (AMV) concentration on (S/N) response function.
Variables concentrations of Precipitator 4-68 mmol/L were synthesis, using 78.5μL
sample volume, distilled water as a carrier stream, 12 mmol/L concentration of CIP.HCl was
injected at injection valve with 4.4 ml/min flow rate to the carrier stream moreover
Precipitator were used. It was observed (figure 2) that an increase in AMV concentration up to
20mmol/L causes a rise in diverge light in terms of peak height t It is probable that this will
enhance the divergence of light toward the solar cells. An excess of AMV more than 20mmol/L
causes a considerably greater effect of agglomeration development of nuclei in a short period of
time, preventing effective diverged light from reaching the detector. The total obtained output
were summed up in table 1-A. Slope-intercept method of choice for the optimum concentration
that will be used as a reagent will be regarded as the best way for the selection of AMV
concentration. Four segmentation were used as shown in figuer 2. A Plot of Y Ƶi(mV) versus
reagent concentration was based on real chart recorded of some selected concentration-profile.
Table 1-B shows the r, a and b value. The researcher found that segment no.2 will satisfy the
request of high sensitivity and high responses profile. The choice will be in the region of 20-28-
40 mmol/L i.e., 20mmol/L →40mmol/L region which represent highest intercept (sensitivity)
and 20mmol/L was the choice which was within the chosen segment. This will give much more
segment for much more accurate choice than choosing the apex of prepared solution. The
researcher will use moving average instead of the apex point prepared by the chemist.
Table 1-A: AMV concentration affects the evaluation of (S/N) energy transducer responses,
which are essential to determining the CIP.HCI.
[NH4VO3] ῩƵi (mV) average RSD Confidence interval at 95%ῩƵi (mV)
mmol/L (n=3) % ± t 0.05/2,n-1σn-1/√ n

4 64 1.89 64 ± 3.006
12 480 0.44 480 ± 5.217
20 792 0.12 792 ± 2.435
28 744 0.16 744 ± 3.006
40 736 0.16 736 ± 3.006
60 728 0.18 728 ± 3.329
68 672 0.18 672±3.900
t0.05/2,2=4.303, ῩƵi (mV): This table gives the (S/N) response expressed as the mean peak
height (n = 3) in mV

3
 Table 1- B: A slope-intercept segmentation pattern is used to select the optimum segment of
concentration (i.e., a3-a5).
No. of Range of [NH4VO3] Segment Slope Intercept r*
segment mmol/L mV/mmol.L-1 mV

1 4-20 a1 – a3 45 -100 0.9966

2 20-40 a3 – a5 -2 834 0.8746
3 40-68 a5-a7 -1 819 0.7954
*r: Correlation coefficient

Figure 2: The effect of different AMV concentrations on the height of ῩƵi (mV): (S/N) energy
transducer response (n=3) in mV, and three data points as one segment their interaction and
selection.
Affect of different salts and acids as a carrier stream on diverge light response ± 90 ° .
The reaction of CIP.HCI (12mmol/L) with AMV (20mmol/L) was studied in different
media of salts and acid (50mmol/L) (Na 2CO3, CH3COONH4, KCl, NaCl, NH4Cl and Na2SO4,
Tartaric acids (C4H6O6), CH3COOH, HCl, H2SO4 and HNO3) solution as a carrier stream
instead distilled water. Figure 3 shows that different salt and acid solutions cause a decrease in
S/N response when compared to D.W; There may be more vacant spaces in between
agglomerates of particulates due to its effect on increasing agglomeration and compactness
rather than increasing the intensity of light transmitted through the particulate. On this premise,
the salts and acids were cancelled throughout the project, making distilled water the best carrier
stream.

Figuer.3: TA comparison of the effects of various salts and acids on the height of height of ῩƵi
(mV): (S/N) energy transducer response (n=3) in mV.
4
Effect of physical parameters
Effect of flow rate
Variation of flow rates ranging from 1ml/min to 7.2 ml/min for each line were used for
CIP.HCI (12mmol/L)-AMV(20mmol/L) system, 78.5µL and open valve mode. Amount of
fluid that will be pumped out to the manifold system .In Figure4, dispersion and dilution
improve at low flow rates, resulting in an increase in *t B (base width of the profile) and
broadening of response maxima. While at higher speed leading to regular profile and very
sharp maxima specifically at 3.2 ml/min . Therefore, a flow rate 3.2 ml/min was used for both
line (reagent stream and carrier stream) as a compromise to obtain narrower Δt B, minimize the
consumption of reactant solution. Table2 tabulate the slope-intercept method and its
segmentation. shows the segmentation sectors and how to use range of values instead single
point reading. A region between 3.2 up to 5.4 ml/min is the choosed segment of optimum
working condition and 3.2ml/min was chosen.

Figure 4: Effect of flow rate on the height of ῩƵi (mV): (S/N) energy transducer response(n=3)
in mV . and three data point as one segment their interaction and selected.

Table 2: Calculate the slope-intercept of a segmentation pattern to identify the optimal segment
for flow rate (i.e.; a4-a6)
No. of Flow rate Segment Slope Intercept r
-1
segment range (ml/min) mV/ml.min mv
1 1-2.5 a1 – a3 -261 1389 0.9952
2 3.2-5.4 a4-a6 -35 930 0.9265
3 5.4-7.2 a6-a8 -98 1273 0.9105
r: Correlation coefficient

Effect of sample volume Using CIP.HCI (12mmol/L)-AMV(20mmol/L) system; the

sample volume was studied. Six variable lengths of Teflon tube of inside diameter of 1mm
were used. A suitable Teflon tubes were cut to have different volumes ranging
40,78.5,135,150,200 and 281µL. Using optimum of flow rate at 3.2ml/min and 60 sec(10mm)
scanning rate of chart recorder and 2V (80mV)-output recorder. The following table 3-A
tabulate the volume and output voltage of signal. A choice is made on the basis of lower
acceptable slope and r due to extended linearity plus high intercept. Taking into account other
consideration e.g. precipitation reaction, as large sample volume will might in some cases
5
blocks the narrow tube diameter, a choice was made for segment 3 i.e., range of 150→281µL.
150 µL was selected as the ideal sample as summed up in table 3-B.
Table 3- A: Effect of sampling volume variations and corresponding measurement output with
CIP.HCI (12mmol/L) and AMV(20mmol/L) systems.
ῩƵi Confidence ΔtB Vadd Concentration t*

D.F at flow cell

(mV) interval at Sec ml mmol/L
sec
Sample
Length

volume μl
average 95%ῩƵi (mV) at at flow cell

RSD
cm

%
(n=3) ± t 0.05/2,n-1σn-1/ flow
√n cell

5.1 40 616 0.19 616±3.006 18 2.060 0.815 14.71 17.22

10.0 78.5 808 0.13 808±2.708 17.7 2.124 0.803 14.94 17.7

17.2 135 976 0.13 976±3.205 18.1 1.991 0.791 15.17 18

19.12 150 1200 0.09 1200±2.857 20.4 2.316 0.725 16.55 19.2

25.47 200 1072 0.10 1072±2.708 20.8 2.487 0.965 12.43 19.6

35.8 281 1064 0.10 1064±2.559 21 2.38 0.76 17.00 19.8

* : Time it takes for the injection valve to reach the measurement cell (in seconds), Δt B: The width of the base of the
peak (Sec), t0.05/2,2=4.303, ῩƵi (mV): (S/N) The average peak heights of energy transducer responses (n=3) in mV . D.F :

Table 3.B: Segmentation pattern slope-intercept to select the optimum segment of sample
volume (i.e.; a4-a6).
No. of segment Range of Sample volume µL segment Slope mV/µL Intercept mV r
1 40-135 a1 – a3 3 484 0.989
2 135--200 a 3- a 5 0.4 1008 0.1394
3 150-281 a4 – a6 -0.9 1311 0.8213
r: Correlation coefficient

Effect of reaction coil expressed as a volume (in microliter).

Variable delay reaction coils (0-235.5µL) were used for this study, which was carried out
on the CIP.HCI (12mmol/L)- AMV(20mmol/L) system. In most cases, adding more length to
the flow gram in CFIA will improve dispersion through a variety of processes (e.g. convection
or diffusion). Reaction coil was attached after Y-junction point in the manifold design as show
in figure 1. The profile and effect of delay reaction coil on sensitivity expressed as an S/N
energy transducer response which is presented in figure 5 From the result of each reaction coil
With a variable volume reaction coil (varying length of the coil), dilution of the sample
segment reaction product will be introduced leading to an even distribution, where with a high
volume, precipitate in a highly dispersed pattern causes a weaker signal. moreover, there was a
decrease in sensitivity with an increase in coil length, which might be due to This might be
connected to the generation of small particles and distributing them over a larger surface area,
making them impossible to detect, i.e., spectrum filtering; the effect of the presence of tiny
particles is abolished since the number of particles is dispersed over a vast volume. As a result,
delayed reaction coils were not used in this study.

6
Fig 5: Effect of mixing coil length on theesponse profile Y Ƶi (mV)-tmin (dmm). CIP.HCI
(12mmol/L)-AMV(20mmol/L)system.
- Estimation of linear dynamic range based on scatter plot variety of Ciprofloxacin.HCl.
via (S/N) .
Using ideal chemical moreover physical factor accomplished in previous sections
AMV (20mmol/L) as a precipitant agent, distilled water as a carrier stream, 150µL- open
valve mode sample volume, 3.2ml/min flow rate for each line and without coil. A series
of solutions for CIP.HCI were prepared in order to study calibration graph representing
by linear equation for scatter plot range and linear range. Ŷ i=a±sat+b±sbt [CIP.HCI]
mmol/L [17,18]. Additionally, the correlation coefficient r, Coefficient of determination
r2, % capital R-squared R2% and t-value had studied. Not only for the new developed
methodology but also for available classical methods (spectrophotometric). Therefore,
made a comparison between newly method and literature cited methods. A series of
solutions for [CIP.HCI] (0.01-50 mmol/L(n=20)) were prepared for the assessment by
ISNAG-Fluorimeter. All estimation was reduplicate three times. All of (S/N) response
(output) of the peak height (mV) was plotted versus the CIP.HCI concentration. An
increase in CIP.HCI concentration leads to an increase in peak height response due to
regular increase of precipitated particle plus its convenient movement with the optimum
flow rate used which in turn to increase of diverged light (as an illustration, deals with the
CIP.HCI concentration and the S/N energy transducer response being exactly
proportional). The correlation coefficient r =0.98623, coefficient of determination r 2 =
0.97.26264, and percent capital R-squared R 2=97.26 percent were obtained from a scatter
plot range for a trial concentration (0.01- 50 mmol/L(n=20). As a result, in order to
enhance the assessment mathematical formulation, a shorter range (0.01-40 mmol/L
(n=19) should be used to increase the correlation coefficient r=0.99390 and percent
capital R-squared R2= 98.78 percent should be employed, as shown in table 4. Similarly,
the UV-spectrophotometric approach, which is based on measurements of absorbance in
the UV-Region for a variable concentration range of 0.001-0.1 mmol/L, is based on
observations of absorbance in the UV-Region.at λmax=272nm (figure 6) [19, 20]. The
correlation coefficient for the range (0.001-0.1 mmol/L(n=14) was r =0.98616, the
coefficient of determination was r2 =0.97251, and the percent capital R-squared R2
7
percent =97.25. A narrower range was used (0.001-0.09mmol/L(n=13)). As a result, the
best linear range is obtained, with a correlation coefficient of 0.99056 and a percent
capital R-squared of 98.12%. In the end, each method's t-calculate exceeds t tab (i.e., t-
value > ttab), suggesting that the linearity versus non-linearity is acceptable. In tablet 4, all
data is summarized.
Figure6. Absorbance UV-spectra of CIP.HCI standard solutions dissolved in water

against distill water as a reference by UV-spectrophotometer (Shimadzu 1800 (classical

method)).
Table 4: Summarizing the results of the various range regressions using the first degree
equations of linear Ŷ =a+bx with Ciprofloxacin.HCl concentration at the ideal condition.
Range of Ŷi = a ± sat+ b ±sbt [CIP.HCI] mmol.L-1 r ttab at Calculated
[CIP.HCI] at confidence level 95%, n-2 r2 95 % , t-value
Type of
mode
mmol/L (n ) R2% n-2 |r| √2−2
√1−r 2
ISNAG-Fluorimeter is used for developing the method.

UV. Sp Traditional absorbance measurement method at λmax= 272 nm

Scatter 0.01-50 (20) 203.632±127.310+75.058±6.234 0.98623 2.101 25.298

plot [CIP.HCL] mmol/L 0.97264
97.26
0.001-0.1 (14) 0.096±0.108+21.295±2.252 [CIP.HCL] 0.98616 2.179 20.602
mmol/L 0.97251
97.25
Linear 0.01-40 (19) 141.289±81.750+82.068±4.662 [CIP.HCL] 0.99390 2.110 37.142
range mmol/L 0.98783
or Liner 98.78
dynamic
range 0.001-0.09 (13) 0.066±0.192+22.895±2.103 0.99056 2.201 23.966
[CIP.HCI]mmol/L 0.98121
98.12
Ŷi: Estimated response(n=3) in mV for modern technique and without unit for uv-spectrophotometric. r:
Correlation coefficient, r2:Coefficient of determination, R2:%capital R-squared, R2= explain variation / total
variation, n:no.of measurements, ttab.=t0.05/2,n-2.

Detection limit (L.D).

A study was achieved to estimate the limit of detection of CIP.HCI by three variation
methods under optimum condition, which tabulated in table 5 .
1. Practically approach: depended on gradual dilution of the lowest concentration in
scatter plot (Using Newly developed method ISNAG-Fluorimeter (4 μmol/L) and UV-
spectrophotometric method as a classical method (0.8μmol/L).

8
 2. Theoretically approach:
A. Depend on slope method and based on the dynamic range for newly developed
method.
B. Depend on the dynamic range or linear range due to has low value of residual (S y/x)
which that equal to Sb of the form Ŷ=Yb+ 3 Sb. Yb [19],[20]
Table 5: Detection limit of CIP.HCI using 150µL and three way.
Practically depended on the progressive dilution for the minimum Theoretical (slope Theoretically (linear
concentration in calibration curve method) depend on equation) depend on
Newly developed method Classical method the value of slope the value of
(4) µmol /L (ῩƵi (mV) UV– spectrophotometric X= 3SB/slope Ŷ= Yb +3Sb
(0.8) µmol/L (Absorbance )
0.221 µg/sample 32 mV 0.294 µg/sample 0.0456 1.371 µg/sample 233.370 µg/sample
SB: Standard deviation of blank for 13 times, Yb: average response for blank= intercept (a), Sb: Standard deviation
equal to Sy/x (residual),

Repeatability
The repeatability and efficiency of ISNAG-Fluorimeter readings, which are equivalent to the
relative standard deviation represented as a percentage using a resonant mercury lamp, were
investigated at a constant Ciprofloxacin concentration. In optimal parameters, HCl was used at
two concentrations (10 and 35 mmol/L). Table 6 summarizes the results of a repetition of
measurements for eight consecutive injections as estimated and the output achieved, with the
highest relative standard deviation (RSD percent) less than 0.34 percent.
Table 6: Repeatability results of CIP.HCI
[CIP.HCI] ῩƵi (mV) RSD % Confidence interval 95% at
mmol/L average (n=8) ῩƵi (mV) ± t 0.05/2,7 σ n-1/√ n
10 976 0.33 976±2.717
35 3040 0.13 3040±3.328
ῩƵi (mV): S/N as an mV energy transducer response expressed, t0.05/2,7=2.365, n= number of injections.

Comparison between two using different methods of analysis CIP.HCI i.e., ISNAG-
Fluorimeter versus UV-spectrophotometric measurement.
In an attempt to establish a comparison between the new method (ISNAG-Fluorimeter) and
traditional method (UV-spectrophotometric) through applying the same series of CIP.HCI
concentration in both methods. When making such a comparison, the primary concern will be
to determine, which is more sensitive than the other and dose the new method give a results that
are significantly higher or lower than the traditional method. When the two methods are to be
compared at corresponding analyte concentration, a results were summarized in table 7. This
idea was applied for individual untreated data to find the linear regression of developed method
using AMV (20mmol/L), 150µL- sample volume open valve mode, 3.2 ml/L flow rate for each
line and without coil, and traditional method, which both have a range from 0.01– 0.1 mmol.L -
1
. The linear regression have a slope value 2039.706 mV/mmol.L -1,while the linear regression
plot for the UV-spectrophotometric method using the same range of concentration, the curve
have slope of 19.772 absorbance/mmol.L-1.
If it assumed that Y1 is the slope (2039.706 mV /mmol.L -1) of the linear regression plot of the
ISNAG-Fluorimeter method.
Which is equivalent = Response1/ concentration ……………………..1
And if it is assumed that Y2 is the slope (19.772 absorbance /mmol.L-1 ) of the linear
regression plot of the UV-spectrophotometric method
Which is equivalent = Response2/ concentration ……………………..2
Divide equation no.1 by equation no. 2
9
The result is = R1/R2 ……………………………………………………3
=2039.706/19.772 = 103.153
A slope ratio shows the value of 103.153 fold ISNAG-Fluorimeter is more sensitive than UV-
spectrophotometric method.
Table 7: Analysis of two methods demonstrating the effects of CPH-HCl concentration on the
response of the (S/N) energy transducer (n=3) in mV by ISNAG-Fluorimeter analyzer and
absorbance without uint by UV– spectrophotometric
ISNAG-Fluorimeter analyzer development UV– spectrophotometric
[CIP.HCI] mmol/L method Classical method

ῩƵi (mV) average (n=3) Absorbance at λmax =272 nm

0.01 32 0.271
0.02 45 0.520
0.03 80 0.768
0.04 108 1.040
0.05 137 1.300
0.06 163 1.560
0.07 170 1.780
0.09 193 1.890
0.10 210 1.990

In addition when using another idea ,i.e: One axis of the regression graph is used for the
responses obtained by new method while the other axis is used for the results obtained by
applying the reference or comparison method to the same concentration. Each point on the
graph represent a single sample analyzed by two separate methods. Applying all this to the data
in table 7, assuming that the X-axis is the response of the UV-spectrophotometric method while
the Y-axis is for the ISNAG-Fluorimeter method. Fig.7 shows the plot of ISNAG-Fluorimeter
method response(R1) in mV (Y-axis ) vs. UV-spectrophotometric method response(R2) (X-
axis) it gives a slope 102.350 mV/absorbance. So that ISNAG-Fluorimeter than UV-
spectrophotometric method more sensitive according to slope ratio. It indicate that the response
of R1 (i.e; ISNAG-Fluorimeter) is more sensitive assuming zero intercept. At non zero intercept
one of the methods is more sensitive from the other method by constant value of concentration.

Figure 7: Calibration graph for the variation of CIP-HCl concentration represented by (S/N)
energy transducer response (n=3) in mV by diverged of incident light vs. Absorbance UV-
spectra
Applications of the ISNAG-Fluorimeter for determining different drug concentrations.
Four different companies of drugs ( AL-jazeera-750mg-Iraq, Micro labs limited -500mg-
India, citro pharma Inc-500mg- Canada and pioneer-500mg -Iraq) were injected on 20 mmol/L
of ammonium metavanadate as precipitation reagent while distilled water as a carrier stream
10
through a two line manifold design system which coupled with a homemade ISNAG-
Fluorimeter analyzer for measure of diverged light of low pressure mercury lamp and this was
compared with the classical UV- spectrophotometric method using maximum wavelength
absorbance at 272 nm [17,18]. Medications were made by transferring 1 mL (50 mmol/L) to
five volumetric flasks (10 mL), then adding 0, 2, 3, 4, and 5 mL from a standard solution of
CIP.HCI (12 mmol/L) to achieve a concentration range of 0, 2.4, 3.6, 4.8, and 6 mmol/L (i.e.,0-
6 mmol.L-1) When CFIA used the identical method of transferring 0.5ml of 0.1mmol/L drug
concentration (variant manufacturing) to all volumetric flasks, followed by 0, 0.1, 0.2, 0.3, and
0.4 ml of 0.1mmol/L to get 0, 0.01,0.01, 0.02, 0.03 and 0.04 mmol/L for Uv-
spectrophotometric analysis (UV-1800 Shimadzu). The data was mathematically adjusted for
standard additions before being compiled into a table 8-A at confidence interval 96% in
addition to practically content of ciprofloxacin.HCI in each sample of drug using two different
methods and efficiency of determination. As well as a comparison treatment of date was
subjected at two different paths. The results were summed in table 8.B.
Individual t-test comparing stated and real value.
A hypothesis can be evaluated in the following way: [21]
There is no substantial difference between the (w i) and the stated value (µ), according to the
null hypothesis.i.e.; Ho: µ = wi
Against, Alternative hypothesis: there may be a vast distinction withinside the quantity of
ciprofloxacin.HCI withinside the specific corporations with claimed value, i.e.; H 1: µ ≠ wi
Since; the value obtained of CIP.HCI- pioneer -Iraq company t cal /-5.136/>> ttab (4.303) which
mean that a The alternative hypothesis will be accepted if the null hypothesis is rejected,
indicating that there is a substantial discrepancy between the stated and measured values.
While, it was inferred that there was no significant difference between the measured value of
ISNAG-Fluorimeter and the claimed value due to t cal. (/-3.464/, 4.052 and /-0.648/) less than t tab
(4.303) by the manufacturer companies of AL-jazeera -Iraq, Micro labs limited – India and citro
pharma Inc- Canada respectively.
In the second test, paired t-tests were used with a significance level of α =0.05 to compare the
developed method using the ISNAG Fluorimeter analyzer with the classical method employing
the Shimadzu (UV-1800 double beam) spectrophotometer.
Assumption
Null hypothesis Ho: μ ISNAG- Fluorimeter =μ UV-sp or μ ISNAG- Fluorimeter -μ UV-sp = zero
There is no discernible difference in the mean of the two approaches.
An alternate theory is that the mean of the conventional approach and the ISNAG- Fluorimeter
analyzer differ significantly.
i.e.; Alternative H1: μ ISNAG- Fluorimeter ≠ μ UV-SP or µ ISNAG- Fluorimeter -μ UV-sp ≠ zero
The acquired outcomes suggest virtually that there has been no huge distinction among newly
advanced approach and UV- spectrophotometric approach (classical method) at 95% (α=0.05)
confidence level as the calculated t cal (/-0.0381/) is less than ttab (3.182) for the determination of
Ciprofloxacin.HCl.
In addition, the ANOVA-Two way was used for study the effect of different methods and the
active ingredient content in addition to the manufacturing companies for the drug using the
following postulate:
1. Difference in methodology.
2. Difference in each company and the active ingredient.
3. The hypothesis test between rows i.e, different methods as a follows:
H₀: There is no difference between rows (i.e., methods)
H₀:µB.P = µISNAG-Fluorimeter = µ UV-spectrophotometer at λmax = 272nm
While an alternative by hypothesis is that there are a significant difference between the three
methods used for the analysis
H1:µB.P ≠ µISNAG-Fluorimeter ≠ µ UV-spectrophotometer at λmax = 272nm

11
 4. The hypothesis test between columns i.e, if you found different between companies of
drug or active ingredient.
The assumption as follows:
H₀: µ(ciprofloxacin,750mg) =µ(Microflox,500mg)=µ(Citroflox,500mg)=µ(Ciproneer,500)
Given there is no significant difference between used company and active in gradient . While
H1: Alternative hypothesis is a significant difference is available i.e., H 1: µ (ciprofloxacin,750mg) ≠
µ(Microflox,500mg) ≠µ (Citroflox,500mg) ≠µ (Ciproneer,500)
From the obtained results (shown in table 8-B), it was found that H₀ is accepted against H 1 due
to Fcal. (0.175) < Ftab. ( 5.143) (sig > 0.05 i.e. no significant difference ), regarding the
comparison between methods. i.e, there are no significant difference between the means of
assessment used for analysis of Ciprofloxacin.HCI while with regard to the comparison
between different companies and content, it was noted that the value of F cal (133.117) >> Ftab
(4.757 which means that there is significant difference is exist between four different
companies of drugs and content ( sig < 0.05 i.e., a significant difference).

12
Table 8.A: Summary of results by standard additions method for the determination of CIP.HCI from different companies by different methods:- ISNAG-
Fluorimeter method using: CIP.HCI -AMV(20mmol/L) system, 150µL sample volume, 3.2ml/L flow rate for each line and without coil and UV-
spectrometer method.
scientific& Type of method
Commercial Newly developed method using ISNAG-Fluorimeter ( mV)
No. of sample
Name, UV. Sp* Classical method absorbance measurement at λmax= 272 nm
Company Confidence Weight of Theoretical [Ciprofloxacin.HCl] mmol/L Equation of standard addition at 95% for
Content interval Sample content for the for newly developed and Classical methods n-2 r
Country For the average equivalent to active ingredient 0 2.4 3.6 4.8 6 r2
Weight of Table 0.9195gm at95% (mg) Ŷi=a±sat+b±sbt [CIP.HCI] R2%
w i ± 1.96σn-1/√n (50mmol/L) Wi±1.96 σ n-1 /√n 0 0.01 0.02 0.03 0.04 mmol/L
at 95% (g) Of the active
Ingredient (wi)
ciprofloxacin 1.17166 750±6.8409 498 718 838 960 1093 489.297±28.358+98.840±7.191 [CIP.HCI] 0.99922
AL-jazeera 0.95568±0.0087 mmol/L 0.99843
CIP.HCI 99.84
1 750mg 0.108 0.360 0.601 0.805 1.070 0.115±0.0389+23.690±1.587 [CIP.HCI] 0.99934
Iraq mmol/L 0.99871
99.87
2 ciprofloxacin 0.77807±0.0062 1.43087 500±3.9582 460 670 779 880 1010 455.243±25.422+90.642±6.447 [CIP.HCI] 0.99251
& Microflox mmol/L 0.99850
Micro labs 99.85
limited 0.140 0.394 0.624 0.945 1.201 0.126±0.061+26.740±2.507 [CIP.HCI] 0.99870
CIP.HCI mmol/L 0.99740
500mg 99.74
India
3 Ciprofloxacin 0.66562±0.00324 1.22407 500±2.4321 499 745 867 990 1114 498.856±1.622+102.421±0.411 [CIP.HCI] 0.98616
& Citroflox mmol/L 0.97251
citro pharma 97.25
Inc 0.121 0.342 0.581 0.797 1.050 0.116±0.025+23.130±1.00941 [CIP.HCI] 0.99972
CIP.HCI mmol/L 0.99944
500mg 99.94
Canada
ciproneer & 0.7556±0.00788 1.38950 500±5.21475 360 520 610 698 796 353.351±2.145+72.455±5.438 [CIP.HCI] 0.99917
Ciprofloxacin mmol/L 0.99833
pioneer 99.83
4 CIP.HCI 0.121 0.341 0.582 0.798 1.023 0.121±0.016+22.610±0.641 [CIP.HCI] 0.99988
500mg mmol/L 0.99976
Iraq 99.98

r: correlation coefficient, r2: coefficient of determination and R2:% capital R-square,R2= explain variation / total variation, t0.025,∞ = 1.96 at 95% , t0.025, 2 = 4.303 for n-1, t0.025, 3 = 3.182.

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Table 8: B: Summary of results for two mode of comparison: Individual t-test (between claimed value and practically value of CIP.HCI) and paired t- test
(between two methods)..
Type of method
Developed method using ISNAG-Fluorimeter ( mV)
UV. Sp* Classical method Absorbance measurement at λmax= 272 nm
Practical concentration Paired t- test compared between
No. of sampte

F-test
(mmol/L) Individual t-test two method
in 10ml For compared between
Practical concentration Weight of ADB in Efficiency of claim value & practical tab at 95%
( mmol/L) tablet determination tcal= Fcal=
value confidence Ftab
in 50 ml w i(mg) ±4.303σ n-1/√ n Rec.%
( W i(mg)-µ ) √ n /σ n-1
W d√ n /σ*n-1
level (n-1)
MSB / MSE
Practical weight of CIP
in ( g)
4.9504
49.5039 742.565±9.235 99.01
0.7426 /-3.4642/≪ 4.303
1
0.0049 The comparison between methods
48.5435 728.153±8.314 97.09
0.7282 0.174909< 5.143253
5.0224
50.224 502.245±2.384 100.45
0.5022 4.0521 << 4.303
0.0047 W d= -0.6335
2
47.1627 471.628±11.782 94.33
0.4716 σ*n-1 =33.22622
4.8707
48.707 487.073±7.532 97.41 n=4
0.4871 /-0.648/ ¿< 4.303
0.0050 /-0.03813/ ≪ 3.182 The comparison between different
3
47.978 499.781±8.315 99.96 companies
0.4998
4.8768 133.1167>> 4.757063
48.768 487.681±0.10.321 97.54
0.4877
/-5.1360/ >> 4.303
0.0053
4
53.4277 534.274±10.632 106.86
0.5343
µ: claim value (mg) =750 and 500 mg, W i: practically weight (n=3),W d: average of different between two methods (developed and classical UV-spectrophotometer) method , ttab
=t0.025,3=3.182 for paired t-test, n( no. of samples ) =4 , σn-1: standard deviation for Individual t-test ,σ*n-1: standard deviation of difference (paired t-test), t0.025,2=4.303 (for Individual t-test
)

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Conclusion: All the results obtained in this whole project shows that these simple instruments (i.e. ISNAG- Fluorimeter which use a long distance of 100
mm length for 2 mm path length . The source for irradiation is a long extended distance for 100 mm and on both sides of 90° angle, each side have four
solar cells. The radiation source is a low pressure mercury lamp which characteristic emitted light: 184.9 nm and predominant at 253.7 nm ) can be used
as a trustful instrument. Solar cells were used in this whole project which was a new approach for using simple electronic circuit , noise free signal

Acknowledgment: The Authors is greatly appreciated to professor Dr. Issam M.A. Shakir . Who without his design and manufacture of all fine details of
this piece of instrument with it simplicity that challenges any available commercial instrument . With deep sincere may God bless his effect to stay as a
leading scientist in an instrumental

15
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