1.2.

INTRODUCTION TO DRUG

SR.
PARAMETER DESCRIPTION
NO.

[(2R)-3,3-Dimethyl-4,4,7-trioxy-4λ6-thia-1-
azabicyclo[3.2.0]heptane-2- carbonyl]oxymethyl(2R)-
Chemical 6-{[(2S)-2-amino-2- phenyl-acetyl]amino}-3,3-
1
name dimethyl-7-oxo-4- thia-1-azabicyclo[3.2.0]heptane-2-
carboxylate

2 Structure

Molecular
3 C
25H30N4O9S2
Formula

4 Category beta-lactamase inhibitors

5 Characteristics
A White-Yellow powder

6 Melting Point 190°C-193°C

Molecular
7 594.7 g/mol
Weight

8 Solubility Soluble in DMSO, Water and Ethanol.

LITERATURE REVIEW

Page 2
1. Literature review

Sr
Title Method Description Ref.
no

1 Cefaclor, Ampicillin, HPLC Column: ODS-2 1

Cefoperazone, Mobile Phase: 0.005 M
Sulbactam, and tetramethylammonium
Sultamicillin Tosylate (TMAH) hydroxide
Are Determined Using Managed pH 3.4 with 1 M
HPLC in Pharmaceutical phosphoric acid
Preparations flow rate: 1 ml / Min
Wavelength: 230 nm

2 HPLC for in-process HPLC Column: (15 cm x 4.6 mm 2

control in the production i.d., 5 um)
of sultamicillin Mobile Phase : 0.005 M
tetramethylammonium
(TMAH) hydroxide
adjusted to pH 3.4 with 1
M phosphoric acid
flow rate : 1 ml / Min
Wavelength : 230 nm

3 Determination of HPLC COLUMN:(1.47 and 4.41 3

sultamicillin in bulkand µg/mL-1 )
phrmaceuticals Mobile phase:4-chloro-7-
preparations via nitrobenzofurazan in pH
derivatization with NBD- 9.0
CL By Flow Rate:60 min
Spectrophotometry. Wavelength:432 nm

4 In vitro evaluation of HPLC Column:7.8% ± 3.1% (n = 4

99mTc‐sultamicillin for
infection imaging
5)
Mobile Phase:0.1% TFA
in H2O (solvent A) and
methanol (solvent B)
Flow Rate:1 ml/min.
Wave length:160 rpm

5 Synthesis of 1,1 UV Solvent: silica gel 5

dioxopenicillanoyloxyme Wave length:237 nm
thyl 6-[d-a
(benzylideneaminophen
ylacetamido)]penicillana
te and analogs. New
intermediates in the
preparation of
sultamicillin

6 The estimation of UV Solvent:ammonium 6

Page 3
 snltamicillin's Wave length:225 nm
pharmacokinetics and
bioavailability via high-
performance liquid
chromatography

7 Prodrugs' growing UV Solvent:formaldehyde 7

significance in today's Wave length:238 nm
medicine development
and design

8 Codrug: A productive UV Solvent:glycolic acid 8

method for medication Wave length:343 nm
enhancement
9 Current Advancements RP-HPLC Column: (150 mm, 4 mm, 9
in Achiral HPLC 4.6 mm i.d.)
Techniques in Fleroxacin and 73: 27
Pharmaceuticals (v/v) make up the mobile
Employing Different phase. Ammonium
Detection Modes phosphate at 50 mM

Flow Rate: 5 minutes

Length of Wave: 300 and
285 nm

10 Mutual prodrugs HPLC Column:ODS column (150 10

containing bio-cleavable mm × 4.6 mm × 5 μm)
and drug releasable Mobile phase:prepared as
disulfide "KH2PO4
linkers solution "(5.82 g in 1000
mL of WFI)": ACN in a
ratio
(80:20, v/v)
Flow rate:1.0 mL/min
Wave length:230 nm

11 Recent advances in UV Solvent:methanol 11

nano-Fenton catalytic Wavelength:202nm
degradation of emerging
pharmaceutical
contaminant

12 Potential alcohol abuse HPLC Column:(2.1 mm · 150 12

and smoking cessation mm, 5 lm)
agents: synthesis and Mobile phase:consisting
hydrolytic behavior of of acetonitrile: 2 mM
two new tripartate ammonium acetate
codrugs of naltrexone Flow rate:0.25 mL/min.
and 6b-naltrexol with
Wavelength:215nm
hydroxybupropion

13 Targeted delivery of UV Solvent:sonicated 13

oxaliplatin via folate- Wave length:181.68nm
decorated niosomal
Page 4
 nanoparticles
potentiates resistance
reversion of colon
cancer cells

14 Benzimidazole UV Solvent:1-alkyl- 14
analogues as efficient benzimidazolium
arsenals in war against Wave length:300nm
methicillin-resistance
staphylococcus aureus
(MRSA) and its SAR
studies

15 High-Performance HPLC Column:C18 column (150 15

Liquid Chromatography– × 2.1 mm, 2.2 µm)
High Resolution Time-of- Mobile phase: 1 mL of the
Flight fil
Mass Spectrometry in trate was discarded, and
the Identification and chromatography was per
formed.
Determination of
Flowrate:30–40 min
Penicillin and
Wave length: 216nm
Tetracycline Group
Antibiotics in

16 Ampicillin and HPLC Column:ODS 2 16

Sulbactam Mobile phase: methanol
Pharmacokinetics and and water in the
Pharmacodynamics in proportion of (60:15:25 )
Continuous Ambulatory Flowrate:5 mL min1
Peritoneal Dialysis Wave length: 218nm
(CAPD)

17 Mutual prodrugs UV Solvent:ibuprofen 17

containing bio-cleavable Wavelength:301nm
and drug releasable
disulfide
linkers

18 synthesis and Potent In UV Solvent:SOCl2 18

vitro Activity of Novel Wavelength:265nm
1H-Benzimidazoles as
Anti-MRSA Agents

19 ANTIBACTERIAL HPLC Column:C1, 4.6 mm × 250 19

PREDRUGS-HPLCFROM mm, 5 µm
1899 TILL 2015 Mobile phase:filtering
through a 0.45 µm
membrane syringe filter
Flowrate:10 min
Wavew length:214nm

Page 5
20 Toxicological UV Solvent:Salmonella 20
evaluation of typhimurium
certain veterinary Wavelength:153nm
drug residues in food

21 Prodrugs Design Based UV Solvent:water molecules 21

on Inter- and Wavelength:285nm
Intramolecular
Chemical Processes

22 Azithromycin's UV Solvent:methanol 22
penetration of middle Wavelength:345nm
ear effusions in children
with acute and secretory
otitis media

23 Synthesis and UV Solvent:chloroform. 23

antimicrobial activity of Wavelength:198nm
novel 2-[4-
(1Hbenzimidazol-1-
yl)phenyl]-1H-
benzimidazoles

24 Producto inesperado de UV Solvent:DMSO 24

la solvatació n de Wavelength:210nm
sultamicilina en DMSO

25 Physicochemical and UV Solvent:water 25

Medicinal Properties of Wavelength:310nm
Tualang, Gelam
and Kelulut sultamicillin:
A Comprehensive
Review

26 Grundlage fü r die UV Solvent:methanol 26

Bewertung “Penicillin- Wavelength:272nm
sensibel” folgende
Dosierungen zugrunde
gelegt

27 ALTERNATIVE UV Solvent:ethanol 27
ANTIMICROBIAL Wavelength:310nm
AGENTS
AGAINST MULTIDRUG-
RESISTANT
GRAM-NEGATIVE
BACTERIA

28 Physicochemical UV Solvent:water,DMSO 28
Characteristics and Wave length:237nm

Page 6
 Bioactive Compounds of
Different mrthod of Uv
and Their Biological and
Therapeutic
Properties: A
Comprehensive Review

29 Effect of Coadministered UV Solvent:aluminium 29

Drugs on the hydroxide
Bioavailability of Wave length:254nm
Cefteram
Pivoxil

30 pHARMACOKINETIC UV Solvent:methanol 30
STUDIES IN ANIMALS OF Wave length:313nm
A NEW PARENTERAL
PENEM CP-65,207 AND
ITS ORAL PRODRUG
ESTER1

31 Historical Review of the UV Solvent:N-alkylthio 31

Penicillin and Related Wave length:300nm
Compounds (β –
Lactams)
Luma Salman Abd

32 genotype of Giardia UV Solvent:Methanol 32

duodenalis in human: Wave length:312nm
first evidence

33 Structural analysis and UV Solvent:CH2Cl2 33

antitussive evaluation of Wave length:290nm
five novel esters of
verticinone
and bile acids

34 Physicochemical and UV Solvent:ethanol 34

Medicinal Properties of Wave length:272nm
Tualang, Gelam
and Kelulut Honeys: A
Comprehensive Review

35 Squamous Cell UV Solvent:methanol 35

Carcinoma in a Capybara Wave length:245nm
(Hydrochoerus
hydrochaeris)

36 Department of UV Solvent:wastewater 36
Genitourinary Medicine, Wavelength:436nm
Royal Infirmary, Edinburgh
EH3 9YW, Laurenston

Page 7
 Place. To be published, all
writers must sign a
permission form.
37 chimica e tecnologie
farmaceutiche,
tossicologiche e
nutraceutico-alimentari
38 The RP-HPLC technique
for determining
methocarbamol,
paracetamol, and related
substances has been
validated for stability.
39 HPLC assay of
acetylsalicylic acid,
paracetamol, caffeine
and
phenobarbital in tablets
40 RP-HPLC-PDA Method
Development and
Validation for Tramadol
Hydrochloride, Caffeine,
and Paracetamol
Determination in
Pharmaceutical
Formulations
41 Use of Stability
Indicating HPLC Method
for Etoricoxib and
Paracetamol
Quantitative
Measurement in
Pharmaceutical Dosage
Form
42 Determination of
Paracetamol and
Tramadol Hydrochloride
in Pharmaceutical
Mixture Using HPLC and
GC–MS
43 Rapid and Simultaneous
Determination of
UV Solvent:1-ethyl-3-(3- 37
dimethylaminopropyl)
Wave length:365nm
RP-HPLC Column:ODS Column 38
Mobile phase:0.05 M
KH2PO4 buffer :
acetonitrile (72.5 : 27.5,
v/v, pH ¼
Flowrate: 1 mL min1
Wave length:225 nm
HPLC Column:C18, 5 mm, 39
250/4.6 mm
Mobile phase:Rheodine
71 25 injector and
Bio Rad 18 01
Flowrate:2.0 ml min1
Wave length:207nm
HPLC Column:C18 column, 4.6 40
mm × 250 mm, 5 µm
Mobile phase:filtering
through a 0.45 µm
membrane syringe filter
to
prevent possible
contamination from
entering
Flowrate:15 min
Wavew length:210nm
HPLC Column:5µm, 250mm X 41
4.6mm
Mobile phase:consisting
of acetonitrile, Water and
methanol in the
proportion of 60:15:25
Flowrate:1.0 mL min-1
Wave length:236nm
HPLC Column:5 µm, 150 × 4.6 42
mm i.d., ThermoHypersil
Keystone, Bellefonte, PA)
Mobile phase:pH (± 0.2
pH units), and
organic strength (± 5%)
Flowrate:1.0
mL/min
Wavelength:220 nm
HPLC Column:10 mm 43
dimethyloctadecyl
Page 8
 Acetylsalicylic Acid, bonded amorphous silica
Paracetamol, and Their (300 mm × 3.9 mm).
Degradation and Toxic Mobile phase:mixture of
Impurity Products methanol:
by HPLC in water (35:65; v/v)
Pharmaceutical Dosaga adjusted to pH 3.1 with
10%
forms
orthophosphoric acid.
Flowrate:1.8 ml.min-1
Wavelength:235 nm
44 RP-HPLC Technique for RP-HPLC Column:ODS, 5µ, C8-3 44
Estimating Etoricoxib Mobile
and Paracetamol from phase:acetonitrile:
Bulk and Tablets phosphate buffer pH
Simultaneously 3.5 (40:20:40 v/v)
Flowrate:6.12 min
Wavelength:242nm
45 Validation and RP-HPLC Column:BDS C18 column 45
Comparison of Cefaclor's (5 μm) x 250 mm x 4.6
Powder for Oral mm
Suspension Dosage Mobile phase:phosphoric
Forms in In-vitro acid (78:10:22 v/v)
Dissolution Studies Flowrate:15 min
Wavelength:265 nm
46 Three UV Solvent:HCl 46
Spectrophotometric Wave Length:207.4nm
Techniques for the
Concurrent
Measurement of
Dicloxacillin and
Ampicillin in the
Presence of Their
Principal Impurity 6.
Acetaminophenolic Acid
47 Pharmacokinetics and UV Solvent:ampicillin 47
bactericidal activity of Wave length:220nm
sultamicillin in infants
and children

48 A double-blinded UV Solvent:penicillin 48
comparative study of Wave length:207 nm
sultamicillin and
potassium penicillin
V in the treatment of
childhood streptococcal
pharyngitis*

49 sultamicillin in the UV Solvent:methanol 49

Treatment of Superficial Wave length:286nm
Skin and Soft Tissue
Infections.

50 Study on Preparation UV Solvent:ethanol 50

Page 9
and Stability of Wave length:205nm
Sultamicillin Tosilate-β-
Cyclodextrin

Page 10
 AIM & OBJECTIVE

2. AIM & OBJECTIVE

3.1 Aim of Present Work

“Development and verification of UV techniques for Quantification Of Sultamicillin using the

hydrotopic solubilization approach”

Page 11
3.2 Objective of Present Work:

 To develop analytical methods for the quantification and characterization of

Sultamicillin utilizing the hydrotopic solubilization approach.

 To develop UV method.

 To systematically optimize hydrotopic solubilization parameters, including the

concentration of hydrotopic agents, temperature, and time, to achieve maximum
solubility of Sultamicillin.

 To conduct a comprehensive validation of the established analytical techniques,

guaranteeing ICH-recommended validation parameters Q2 (R1) guidelines.

Page 12
 RATIONAL

 4. The literature assessment found that no analytical techniques were provided for
determining Sultamicillin in bulk and their dosage forms using the hydrotopic
solubilization approach.

 Hence it was thought of interest to develop an analytical method (UV

spectroscopy) for determination of Sultamicillin in bulk and their dosage forms
Page 13
using the hydrotopic solubilization approach and to validate this method using
UV-Visible Spectroscopy.

Page 14
 MATERIALS AND METHOD

 Analytical method development using hydrotopic solubilization approach

5.1 Chemicals

 Standard API of Sultamicillin drug.

 Hydrotopic solubilizing agent & Solvent:

Page 15
Methanol, Acetonitrile, Ethanol,

DMSO, Phosphate Buffer, Urea, Citrate of sodium, Sodium Benzoate, and Sodium
Salicylate, Niacinamide, Sodium Acetate, Water.

5.2 Instruments
 UV-Visible spectrophotometer

 Ultra Sonicator

 FTIR

 Analytical Balance

 pH Meter

5.3 Authentication of API

 Infrared Spectroscopy
 Solubility
 Melting Point

Page 16
 EXPERIMENTAL WORK

• To obtain API and Chemicals

• Method Development

• Method Validation

• Data Compilation

6.1 To obtain API and Chemicals

Page 17
 Melting point determination

 Sultamicillin's melting point has been established using open capillary techniques,
which have yielded the same result.

 The drug’s melting point was recorded and compared with literature values. The
melting point of Sultamicillin was found to be 190-192°C.

Reported Melting point Merely noted melting

Drug point

Sultamicillin Tosylate 191° C 190-192°C

6.2 Instruments of Work

 UV-Visible spectrophotometer

 Ultra Sonicator

 FTIR

 Analytical Balance

 pH Meter

6.3 Chemicals Use

 Standard API of Sultamicillin drug.

 Hydrotopic solubilizing agent & Solvent:

o Methanol, Acetonitrile, Ethanol,

o DMSO, Urea, Sodium Benzoate, Sodium Citrate, Sodium Salicylate, Polyethylene

Glycol, Niacinamide, Sodium Acetate, Water

6.4 Authentication of API

 Infrared Spectroscopy

 Solubility

 Melting Point

Page 18
6.5 IR spectra interpretation of Sultamicillin

6.6 IR spectra interpretation of Sultamicillin

Page 19
 Functional Group Wave Number (cm-1)
Sr No.

1. C-H (alkene) 3009.10 cm-1

2. C=O (amide) 1658.65 cm-1

3. N-H (amine) 1630.15 cm-1

4. C=C (benzene) 1430.24 cm-1

5. C-N (amine) 1223.68 cm-1

6. C-C (alkane) 1011.71 cm-1

7 S=O(Sulphoxide) 1030-1060cm-1

7. Validation Parameters
• Calibration Curve

• Linearity

• Accuracy

• Precision

• Robustness

• Ruggedness

Page 20
 • LOD and LOQ

• Assay

7.1 The linear nature

An analytical method is considered linear if it provides test results that are directly, or
through a precise mathematical translation, proportionate to the analyte concentration
in samples falling under a specified range. It is possible to establish linearity for all
predicted contaminants, preservatives, and active ingredients. Standards are used for
evaluation.

Plotting absorbance (Y) versus concentration (X) creates a linear curve with a
correlation equation, which is used to create a calibration curve.

Y = mX + C where Slope C = Y = m Intercept “M" and "c" can be stated mathematically as

𝑵 ∑ 𝐱𝟏𝐲𝟏 - ∑ 𝐱𝟏 ∑ 𝐲𝟏
𝒎=
𝑵 ∑𝒙𝟏 𝟐 (∑ 𝒙𝟏)𝟐

𝑥̅1 = Average of all the values of X c = 𝒚 𝟏 - 𝒙𝟏

𝑦1 = Average of all the values of Y

r=
𝑵 ∑ 𝐱𝟏𝐲𝟏- ∑ 𝐱𝟏 ∑ 𝐲𝟏
√[𝑵 ∑𝐱𝟏 𝟐 - ∑ 𝒙𝟏] - [𝑵 ∑𝐲𝟏 𝟐 -∑ 𝒚𝟏]

The values of r must fall between +1 and -1; the more closely the r value approaches 1
or -1, the more linear the procedure will be.
• Ideally, the coefficient of determination (r2) is higher than 0.998.
• The residuals Lot S30 P30 should not contain any curvature.
• It is crucial that the Y-intercept stays close to zero at 36 t.

7.2 Linearity and calibration graph

 Preparation of Standard Stock Solution

Ten milligrams of the antibiotic, precisely weighed, were added to a ten-milliliter

volumetric flask along with ten milliliters of 2 M urea as a hydrotropic agent .

The flask sonicated for about 15 min to solubilize the drug. Concentration is 1000 μg/mL
of stock solution.

Page 21
  Preparation of Working Solution

The standard stock solution (1000 μg/mL) was further diluted with distilled water to
obtain 2, 4, 6, 8, 10μg /mL solution and absorbance were noted at 220 nm against
distilled water as blank and shown in fig. 2.

Figure 2. Spectra of Sultamicillin in 2 M Urea (2, 4, 6, 8 and 10 μg /mL)

4.405

4.000

3.000
Abs.

2.000

1.000
228 nm.

0.000

-0.453
200.00 250.00 300.00 350.00 400.00
nm.

Table 1. Linearity of Sultamicillin at λmax = 228 nm

Concentration
Rep-1 Rep-2 Rep-3 Rep-4 Rep-5 Mean S.D %RSD
(μg/mL)
2 0.067 0.066 0.066 0.065 0.066 0.066 0.0007 1.06
4 0.084 0.084 0.082 0.082 0.083 0.083 0.001 1.20
6 0.104 0.102 0.102 0.103 0.102 0.102 0.0008 0.784
8 0.122 0.123 0.119 0.122 0.120 0.121 0.001 0.826
10 0.146 0.144 0.144 0.142 0.145 0.144 0.001 0.694
12 0.161 0.163 0.158 0.162 0.162 0.161 0.0019 0.621
*Mean of 5 Replicates at 6 Concentration level Average (%RSD) = 0.864

Table 2. Calibration curve of Sultamicillin

Page 22
 Calibration curve of Sultamicillin at 228 nm in 2 M Urea
0.8

0.7
f(x) = 0.0333 x + 0.3432
R² = 0.995725766623266
0.6
bsorbance
0.5

0.4

0.3

0.2

0.1

Accuracy
0
1 2 3 4 5 6 7 8 9 10 11
Concentration(ppm)

 Make a standard drug solution with a concentration of any drug within the linearity range. (8
μg/mL)

 In order to conduct recovery tests, a known amount of tablet solution (80, 100, 120 % of
the 8 μg /mL concentration) was spiked. At, the absorbance was determined. 228 nm using
UV Spectrophotometry against distilled water as a blank.

 To determine whether the additional drug sample was recovered, the analysis process was
repeated. Three replicates at three different concentration levels underwent this recovery
analysis again.
Table 3. Recovery study of Sultamicillin
Level of
Amount added Total amount
% Concentration %
(spike) in Absorbance recovered
Recover (μg /mL) Recovery
μg /mL (μg /mL)
y
4 3.2 0.118 7.17 99.98
80 4 3.2 0.116 7.30 99.99
4 3.2 0.119 7.39 100.03
4 4 0.219 8.09 100.25
100 4 4 0.222 8.16 100.44
4 4 0.225 8.46 100.64
4 4.8 0.321 9.17 101.58
120 4 4.8 0.325 9.49 101.65
4 4.8 0.322 9.83 100.56

Page 23
 8. Precision
The proximity of a sequence of individual analyte measurements determines the accuracy of
the procedure.

8.1 Types of Precision

1) Repeatability: the outcomes are replicated after a little interval.
2) Intermediate Precision: in a lab, many tools are employed to consistently produce
results.
3) Reproducibility: get consistent results using different lab conditions, different analysts,
and diverse environments.
 Intraday precision is the ability to analyze a sample on the same day.
 Inter-day precision refers to analyzing a sample on a separate day.

Table 5. Result of Intraday Precision

Concentration Absorbanc Standard

Mean* % RSD
(μg /mL) e Deviation*
0.321
8 0.322 99.48 0.5814 0.582
0.331
0.411
10 0.425 99.69 0.8096 0.890
0.438
0.511
12 0.521 99.90 0.9104 0.904
0.564
*Mean of 3 Replicates at 3 Concentration level Average (%RSD) = 0.792

Table 6. Result of Inter day Precision

Page 24
 Concentration Standard
Absorbance Mean* % RSD
(μg /mL) Deviation*
0.323
8 0.324 3.78 0.1024 1.22
0.334
0.413
10 0.427 5.39 0.0916 0.993
0.436
0.513
12 0.525 7.24 0.0842 0.858
0.568
*Mean of 3 Replicates at 3 Concentration leve Average (%RSD) = 1.051

9. Robustness

• An analytical method's robustness is determined by how well it can withstand

slight but intentional changes in its parameters.

• Robustness study was performed by changing in the scanning speed of UV-

visible Spectrophotometer.

• Use distilled water to dilute the stock solution for T45 and prepare a solution
having a final concentration of 10 μg /ml. and scanned in the region 200-400 nm
with the help of UV-visible Spectrophotometer by changing in the scanning speed
i.e., fast, medium and slow.

• Reading were Obtain three times for each factor and then further calculation
were done by taking the mean of each factor.

Table 7. Result of Robustness study

Variation and level Concentratio Absorbance %RSD

Page 25
 n (μg /mL)
8 0.488
Fast 8 0.489 1.11
8 0.495
8 0.478
Changing in
Medium 8 0.465 0.743
scanning speed
8 0.478
8 0.484
Slow 8 0.477 0.491
8 0.486

Table 8. Statistical Validation for Robustness study

Co-efficient of
% Mean Standard
Variation and level Variation* (%
Recovery* Deviation*
R.S.D.)
Fast 100.02 1.131 1.11
Changing in
Medium 99.52 0.746 0.749
scanning speed
Slow 99.76 0.450 0.452
*n = 3

• The percentage R.S.D. is below 2%, as mandated by ICH rules.

10. Ruggedness

Page 26
  The degree of repeatability of test findings acquired by analyzing the same
sample under various situations, such as different analysts, was used to conduct
the ruggedness research.
• The solution of concentration 10 μg/mL of Sultamicillin was prepared by
diluting and preparing the stock and diluted solution by using distilled water.
• Two separate analysts completed the process, and the findings were collated,
and calculations were made to determine the SD, %RSD, and standard error.

Table 9. Result of Ruggedness study

Amount
Concentratio
Variation and level Absorbance recovered % Recovery
n (μg /mL)
(μg /mL)
8 0.488 7.49 0.449
Analyst 1 8 0.489 8.02 100.25

Different 8 0.495 7.13 100.26

analyst 8 0.466 7.42 0.451
Analyst 2 8 0.474 7.10 100.11
8 0.432 8.16 99.14

Table 10. Statistical Validation for Ruggedness study

Co-
efficient
% Mean Standard
Variation and level of Standard Error*
Recovery* Deviation*
Variation*
(% R.S.D.)

Differen Analyst 1 100.02 0.442 0.448 100.02

t analyst Analyst 2 99.76 0.453 0.456 99.76

*n = 3
 The % R.S.D. is less than 2 % as required by ICH guidelines.

11. Limit of Detection (LOD)

 The lower limit of an analyte that can be identified but does not need quantification is
stated as the LOD of an analytical technique.

Page 27
The lowest concentration sample that, given the specified experimental circumstances,
may be identified but not necessarily measured is known as the limit of detection, or
LOD. Typically, it is stated as the analyte concentration in the sample.

By comparing the signal-to-noise ratio of samples with known analyte concentrations

with blank samples, the lowest level at which the analyte may be consistently identified
is established. The limit of detection for instrumental methods is established through
this approach. A 2:1 or 3:1 signal-to-noise ratio is suitable.

By dividing the base peak by the standard deviation of all data points below a
predetermined threshold, one may calculate the signal-to-noise ratio. Limitation of
detection
LOD = 3.3 X 𝑺𝑫
𝑺𝒍𝒐𝒑𝒆 𝒐𝒇 𝒄𝒂𝒍𝒊𝒃𝒓𝒂𝒕𝒊𝒐𝒏 𝒄𝒖𝒓𝒗𝒆

 Table 11. Results of regression equation:

σ
LOD ¿ 3.3 × Equation Intercept Slope
S
Were, Y=0.0096x-0.0204 -0.0324 0.0096

σ = Standard deviation of intercept Y=0.0098x-0.0433 -0.0353 0.0098

S = Slope of calibration curve Y=0.0095x-0.0302 -0.0382 0.0095

Y=0.0098x-0.0339 -0.0379 0.0098
Y=0.0098x-0.0346 -0.0396 0.0098
Mean =
S.D of Intercept = 0.001931
0.0098

Calculation

σ
LOD ¿ 3.3 ×
S
0.00073
LOD ¿ 3.3 ×
0.0034
LOD ¿ 3.3 ×0.214

LOD ¿ 0. 70 6 μg /ml

11.1 Limit of Quantification (LOQ)

Page 28
The limit of quantification, or LOQ, is the lowest concentration of analyte in the sample
that can be determined with a respectable level of accuracy and precision. It is
frequently expressed as the sample's analyte concentration. By making comparisons, it
may be determined.

Compared to the signals from blank samples, signals from samples with known low
analyte concentrations were observed. The lowest analyte concentration at which
precise measurements may be taken is ascertained. A signal-to-noise ratio of 10:1 is
deemed acceptable. Other approaches focus on determining the standard deviation of
the responses and the slope of the calibration curve.

LOQ = 10 X 𝑺𝑫
𝑺𝒍𝒐𝒑𝒆 𝒐𝒇 𝒄𝒂𝒍𝒊𝒃𝒓𝒂𝒕𝒊𝒐𝒏 𝒄𝒖𝒓𝒗𝒆

where SD is the calibration curve intercepts' standard deviation

 LOQ of an analytical method is expressed as lower concentration or limit of analytical that

can be detected and quantified with good accuracy and precision.

σ
LOQ ¿ 10 ×
S
0.001641
LOQ ¿ 10 ×
0.00 34
LOQ ¿ 10 ×0.1877
LOQ ¿ 1.87 μg /mL

12. Assay
The commercially available tablet was analyzed using a recommended technique. The
test findings mostly agreed with the label claim. No typical lubricants, solvents, or
diluents were utilized to obstruct the recommended methods.

examination. The novel spectrophotometry technology is therefore rapid, simple to use,

accurate, precise, and reasonably priced. Tablet dosage forms might be utilized for
regular analysis.

Analyzing Solid Formulation using the Suggested Method. The suitability of the
proposed approach was assessed by an examination of the commercially available
tablet formulation.

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 Table 12. Results of assay study
Co-
Test efficient
Concentratio Standard % Standard
Absorbanc of
n (μg /mL) Absorbance Recovery Deviation*
e Variation*
(% R.S.D.)
0.104 0.102 99.02
0.104 0.101 98.05
0.104 0.102 99.02
1.17 1.175
10 0.104 0.101 100.97
0.104 0.102 99.02
0.104 0.103 100.97
% Mean Recovery* = 99.50
*n = 6

A summary of the suggested method's validation parameters

Sr.
Parameters Values (Sultamicillin)
No.
Linearity Range
1. 02- 10
(μg /mL)
Correlation
2. 0.9957
Coefficient (R²)
% Mean
Accuracy % R.S.D.
Recovery*± S.D.*

3. 80 % 100.56 ± 0.627 0.622

100 % 100.32 ± 0.433 0.430
120 % 100.12 ± 0.325 0.329
Precision (% R.S.D.)
4. Intra Day 0.849
Inter Day 1.015

Sr. Parameters Values (Sultamicillin)

Page 30
No.
Robustness % Mean Recovery*± S.D.* % R.S.D.
Fast 100.02 ± 1.131 1.11
5. Changing in
Medium 99.52 ± 0.746 0.749
scanning speed
Slow 99.76 ± 0.450 0.452
Ruggedness % Mean Recovery*± S.D.* % R.S.D.
6. Different Analyst 1 100.02 ± 0.450 0.448
analyst Analyst 2 99.76 ± 0.450 0.456
7. Limit of Detection – LOD (μg /mL) 0.706
Limit of Quantification – LOQ (μg
8. 1.87
/mL)
Assay (% Mean Recovery*± S.D.*) &
9. 99.50 ± 1.17 1.175
(% R.S.D.)

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