1 . 1 Theory o f U. V.

Spectroscopy:

INTRODUCTION:
Many molecules absorb ultraviolet or visible light. The absorbance of a solution increases as
attenuation of the beam increases. Absorbance is directly proportional to the path length, b, and
the concentration, c, of the absorbing species. Beer's Law states that

A = cbc, where c is a constant of proportionality, called the absorb! ivity.

Different molecules absorb radiation of different wavelengths. An absorption spectrum will show
a number of absorption bands corresponding to structural groups within the molecule. For
example., the absorption that is observed in the UV region for the carbonyl group in acetone is
of the same wavelength as the absorption from the carbonyl group in diethyl ketone.

Electronic transitions: The absorption of UV or visible radiation corresponds to the

excitation of outer electrons. There are three types of electronic transition which can' be
considered;

1. Transitions involving n, a, and n

electrons
2. Transitions involving charge-transfer electrons
3. Transitions involving d and/electrons

When an atom or molecule absorbs energy, electrons are promoted from their ground
state to an excited state. In a molecule, the atoms can rotate and vibrate with respect to
each other. These vibrations and rotations also have discrete energy levels, which can be
considered as being packed on top of each electronic level.

(Figure-1: Vibrational and electronic transitions)

 Absorbing Species Containing
Absorption of ultraviolet and visible radiation in organic molecules is restricted to certain
functional groups (chrornophores) that contain valence electrons of low excitation energy. The
spectrum of a molecule containing these chrornophores is complex. This is because the
superposition of rotational and vibrational transitions on the electronic transitions gives a
combination of overlapping lines. This appears as a continuous absorption band. Possible
electronic transitions of 7c,o , and n electrons are;

Figure --2: Electronic transitions

o—»o Transitions
An electron in a bonding o orbitalis excited to the corresponding antibonding orbital. The
energy required is large. For example, methane (which has only C-H bonds, and can only
undergo c—+o~' transitions) shows an absorbance maximum at 125 run. Absorption maxima
due to a—»a" transitions are not seen in typical UV-Vis. spectra (200 - 700 run

n—*o* Transitions
Saturated compounds containing atoms with lone pairs (non-bonding electrons) are capable of «—
KJ* transitions. These transitions usually need less energy than o—*CJ* transitions. They can be
initiated by light whose wavelength is in the range 150 - 250 nm. The number of organic functional
groups with p—*<J peaks in the UV region is small n—>n and n—>n * Transitions

Most absorption spectroscopy of organic compounds is based oil transitions of n or TT electrons

to the TC" excited state. This is because the absorption peaks for these transitions fall in an
experimentally convenient region of the spectrum (200 - 700 nm). These transitions need an
unsaturated group in the molecule to provide the JI electrons.

Molar absorptivities from n—*n transitions are relatively low, and range from 10 to 100 L mof!
cm'1 %—m transitions normally give molar absorptivities between 1000 and 10.000 L mol"1
cm"1
The solvent in which .the absorbing species is dissolved also has an effect on the spectrum of
the species. Peaks resulting from n transitions are shifted to shorter wavelengths {blue shift)
with increasing solvent polarity. This arises from increased solvation of the lone pair, which
lowers the energy of the n orbital.
Often (but :.oi always), the reverse (i.e. red shift) is seen for n—n transitions. This is caused
by attractive polarisation forces between the solvent and the absorber, which lower the energy
levels of both the excited and unexcited states. This effect is greater for the excited state, and
so the energy difference between the excited and unexcited states is slightly reduced - resulting
in a small red shift. This effect also influences p—*it transitions but is overshadowed by the
blue shift resulting from solvation of lone pairs.

Instrumentation :

The light source:

To get the range of wavelengths from about 200 nm to about 800 nm. a combination of two is
used - a deuterium lamp for the U.V part of the spectrum, and a tungsten I halogen lamp for the
visible pan.

The combined output of these two bulbs is focussed on to a diffraction grating.

The diffraction grating and the slit:

A diffraction grating splits light into its component colors more efficiently.

Ight SOUCE ( Figure-4: Function of a Diffraction grating )

The arrows show the way the various wavelengths of the light are sent off in different directions.
The slit only allows light of a very narrow range of wavelengths through into the rest of the
spectrometer.

By gradually rotating the diffraction grating, you can allow light from the whole spectrum (a tiny
part of the range at a time) through into the rest of the instrument.

The rotating duces:

This is the clever bit! Each disc is made up of a number of different segments. Those in the
machine we are describing have three different sections - other designs may have a different
number.

The light comiog from the diffraction grating and slit will hit the rotating disc and one of three
things can happen.

1. If it hits the transparent section, it will go straight through and pass through the cell
containing the sample. It is then bounced by a mirror onto a second rotating disc.

This disc is rotating such that when the light arrives from tiie first disc, it meets the
mirrored section of the second disc. That bounces it onto the detector.

It is following the red path in the diagram:

 2. If the original beam of light from the slit hits the mirrored section of the first rotating disc,
it is bounced down along the green path. After the mirror, it passes through a reference
cell (more about that later).

Finally the light gets to the second disc which is rotating in such a way that it meets the
transparent section. It goes straight through to the detector.

3. If the light meets the first disc at the black section, it is blocked - and for a very short while
no light passes through the spectrometer. This just allows the computer to make allowance
for any current generated by the detector in the absence of any light.

The sample and reference cells:

These are small rectangular glass or quartz containers. They are often designed so that the light
beam travels a distance of 1 cm through the contents.

The sample cell contains a solution of the substance you are testing - usually very dilute. The
solvent is chosen so that it doesn’t absorb any significant amount of light in the wavelength range
we are interested in (200 - 800 nm).

The reference cell just contains the pure solvent.

The detector and computer:

The detector converts the incoming light into a current. The higher the current, the greater
the intensity of the light.

For each wavelength of light passing through the spectrometer, the intensity of the fight
passing through the reference cell is measured. This is usually referred to as !<, - that’s 1
for Intensity.

The intensity of the light passing through the sample cell is also measured for that
wavelength - given the symbol, I. If I is less than lQ, then obviously the sample has
absorbed some of the light. A simple bit of mathematics is then done in the computer to
convert this into something called the absorbance of the sample - given the symbol, A.

For reasons which will become clearer when we do a bit of theory on another page, the
relationship between A and the two intensities is given by:

On most of the diagrams you will come across, the absorbance ranges from 0 to 1. but it
can go higher than that.

An absorbance of 0 at some wavelength means that no light of that particular wavelength

has been absorbed. The intensities of the sample and reference beam are both the same,
so the ratio VI is I. Log|0 of 1 is zero.

An absorbance of 1 happens when 90% of the light at that wavelength lias been absorbed
- which means that the intensity is 10% of what it would otherwise be.

in that case, \J\ is 100’JO (=10) and log:r, of 1 0 is 1

1.2 Validation of a Stability Indicating Method (SIM); v”*
.Method validation can be defined as (ICR) “”Establishing documented evidence, which provides
a high, degree of assurance that a specific activity will consistently produce a desired result or
product meeting its predetermined specifications and quality characteristics”.

Method validation is an integral part of the method development; it is the process of

demonstrating that analytical procedures are suitable for their intended use and that they support
the identity, quality, purity, and potency of the drug substances and drug products. Simply,
method validation is the process of proving thai an analytical method is acceptable for its intended
purpose.

Method Validation, however, is generally a one-time process performed after the method
has been developed to demonstrate that the method is scientifically sound and that it serves the
intended analytical purpose.

Key analytical parameters include:-

(a) Recovery (g) Limit of quantitation
(b) Response function (h) Ruggedness
(c) Sensitivity (i) Robustness
(d) Precision (j) stability

(e) Accuracy
(f) Limit of detection

(a) Recovery
The absolute recovery of analytical method is measured as the response of a processed spiked
matrix standard expressed as a percentage of the response of pure standard, which has not been
subjected to sample pre-treatment and indicates whether the method . provides a response for the
entire amount of analyte that is present in the sample. It is best established by comparing the
responses of extracted samples at low, medium and high concentrations in replicates of at least 6
with those non-extracted standards, which represent 100% recovery.

response of an analyte spike into matrix (processed)

Absolute recovery = ---------------------------------------------------- X 100
response of analyte of pure standard (unprocessed)
b) Response of function

• In chromatographic methods of analysis, peak area or peak height may be used as response
function to define the linear relationship with concentration known as the calibration model.
It is essential to verify the calibration model selected to ensure that it adequately describes
the relationship between response function (Y) and concenu-ation (X).

(c) Sensitivity

“the method is said to be sensitive if small changes in concentration cause large changes in
response function. The sensitivity of an analytical method is determined from the slope of the
calibration line.

(d) Precision

The purpose of carrying out a determination is to obtain, a valid estimate of a ‘true’ value.
Precision and accuracy together determine the error of an individual determination..

Precision refers to the reproducibility of measurement within a set, that is, to the scatter
of dispersion of a set about its central value. The term ‘set’ is defined as referring to a number
(n) of independent replicate measurements of some property.

(e) Accuracy

Accuracy normally refers to the difference between the mean x****, of the set of results and the
true or correct value for the quantity measured. According TO ITJPAC accuracy relates to the
difference between results (or mean) and the true value

Accuracy is best reported as percentage bias, which is calculated from the expression
1.Calibration

Calibration is the most important step in bioactive compound analysis. A good precision and
accuracy can ordy be obtained when a good calibration procedure is adopted. In the
spectrophotomctric methods, the concentration of a sample cannot be measured directly, but is
determined using another physical measuring quantity ‘y’ (absorbance of a solution). An
unambiguous empirical or theoretical . relationship can be shown between this quantity and the
concentration of an analyte. The calibration betWCm y = g (x) is directly useful and yields by
inversion of the analytical calculation function.

The calibration function can be obtained by fining an adequate mathematical model through the
experimental data. The most convenient calibration function is linear, goes through the origin and
is applicable over a wide dynamic range In practice, however many deviations from the ideal
calibration line may occur. For the majority of analytical techniques the analyst uses the
calibration equation.

Y = a + b.v

In calibration, univariate regression is applied, which means that all observations are dependent
upon a single variable X.

2.Standard deviation of slope (Sb)

The standard deviation of slope is proportional to standard error of estimate and

inversely .proportional to the range and square root of the number of data points.
4.Correlation Coefficient, (r) :
The correlation coefficient r (x,y) is more useful to express the relationship of the chosen scales.
To obtain a correlation coefficient the covariance is divided by the product of the standard
deviation of x and y

5.Linearity and sensitivity of the metliod :

Knowledge of the sensitivity of the color is important and the following terms are
commonly employed for expressing sensitivity. According 10 Bouger- Lambert — Beer’s law.
log intensity of incident-radiations

The absorbance (A) is proportional to the concentration (e) of the absorbing species, if
absorptivity (e) and thickness of the medium (t) ate constant. When c is in moles per liter, the
constant is called molar absorptivity. Beer’s law limits and emax values are expressed as ug ml-
’ and mole-1 cm-1 respectively.

SandelPs sensitivity refers to the number of fig of the drug to be determining, converted
to the colored product, which in a column solution of cross section lcm2 shows an absorbance
of 0.001 (expressed as jig cm-2).

(f) Limit of detection (LOD):

The limit of detection (LOD) of an analytical method may be defined as the concentration,
which gp.es rise to an instrument signal that is significantly different from the blank. For
spectroscopic techniques or other methods that rely upon a calibration curve for quantitative
measurements, the JUPAC approach employs the standard deviation of the intercept (Sa),
which may be related to LOD and the slope of the calibration curve, b, by
 LOD - 3 Sa / b

(g) Limit of quantitation (LOQ):

The LOQ is the concentration that can be quantitate reliably with a specified level of accuracy
and precision. The LOQ represent the concentration of analyte that would yield a signal-to-noise
ratio of 10.

LOQ- 10Sa/b

Where, Sa- the estimate is the standard deviation of the peak area ratio of analyte to IS (5
injections) of the drugs, b -is slope of the corresponding calibration curve.

(h) Ruggedness:

Method Rugged ness is defined as the reproducibility of results when the method is performed
under actual use conditions. This includes different analysts, laboratories, columns, instruments,
source of reagents, chemicals, solvents etc. Method ruggedness may net bo known when a method
is first developed, but insight is obtained during subsequent use of that method.

(^Robustness:

The concept of robustness of an analytical procedure has been defined by the ICH as "a measure
of its capacity to remain unaffected by small but deliberate variations in method parameters ".
The robustness of a method is the ability to remain unaffected by small changes in parameters
such as pH of the mobile phase, temperature, %organic solvent strength and buffer concentration
etc. to determine the robustness of the method experimental conditions were purposely altered
and chromatographic characters were evaluated.

(j)Stability:

To generate reproducible and reliable results, the samples, standards and reagents used :'O:
the UV method must be stable for a reasonable time (e.g. one day. one week, one month
depending upon need).
1.3 STABILITY STUDIES:

In the rational design and evaluation of dosage forms for the drugs, the stability of the activity
components must be a major criterion in determining their stability. The medicine has to reach
the patient id an active and acceptable form maintaining the criteria for acceptable equality. The
quality of the product has to be retained as long as the product is offered for sale or for
administration to the patient. Several forms of instability can lead to the rejection of a drug
product. STABILITY is officially defined as the time lapse during which the drug product retains
the same properties and characteristics that it possessed at the time of manufacture. The stability
of a product is expressed as the expiry period or technically as shelf life.

Significance of stability studies

1.There may be chemical degradation of the active drug, leading to a substantial lowering of the
quality of the therapeutic agent in the dosage form

2.Although chemical degradation of the active drug may not be extensive, a toxic product may
be formed in the decomposition process

3.Instability of a drug product can lead to a decreased in its bioavailability, rather than to ioss oi
drug -.>i to formation of toxic degradation products. This reduction in bioavailability can result
in a substantial lowering in the therapeutic efficacy of the dosage form. This phenomenon can be
caused by physical <>i chemical changes in the excipients in the dosage form, independent of
what ever changes the active ilmy may have undergone

4.There may be substantial changes in the physical appearance of the dosage form. The
physical - h.uip include mottling of tablets, creaming of emulsions and caking of substances,
the paticnl will hi.. I>. n. loose confidence in the drug product which then have to be rejected.

Stability studies are done in two ways

1. Accelerated testing: Defined as studies designed to increase Ihe rate o! chemu.il il< •■ ul.ii
>i
physical change of an active drug substance or drug product using exaggerated shinier - himI
...................................................................................................................................................
of"the formal, definitive, storage program.

2. Stress testing: Defined as the determination of the intrinsic stability of the m..I. nl< !•• li iiinu
'degradation pathways in order to identify the likely degradation product.-; arid u> >ili.l..i. iln-
inl.iur,
indicating power of the analytical procedures used.
An intrinsic stability characteristic of drug molecule include.-, developing
mUi ■»•«...I.t.M . i

JConditions leading to degradation

  Rates of degradation
 Chemical structure of the degradation products
 Degradation pathways.

Stress testing studies involve exposure of the drug substance to the stress conditions of
heat, humidity, photo stress (LTV and VIS), oxidative conditions, and aqueous conditions
across a broad pH range. The intent is to induce 10-20% degradation of the parent drug.
Without stress testing there is no way to assess whether or not the method will resolve and
detect the degradation products. Through stress testing studies need to evaluate the four
main degradation pathways of pharmaceuticals:

1 .Hydrolytic,

2.Thcrmolytic,

3.Phololytic

4,Oxidative.

1. Hydrolytic degradation: To evaluate hydrolytic pathways, aqueous solutions (using 100%

aqueous conditions when solubility permits or adding an inert co-solvent such as acetonitrile) can
be prepared at various pH conditions and samples can be stored at elevated temperatures if no or
slow degradation is observed at room temperature.
2. Thermolytic Degradation: To evaluate thcrmolytie pathways, elevated temperature (e.g., 50-
80°O) ill the solid state and/or in solutions can be used. Above S0°C man)’ compounds begin to
degrade via different mechanisms, giving rise to degradation products. For solid- state stressing,
the use of high and low humidity atmosphere at the elevated temperatures is appropriate.
3. Bhotolytic Degradation: The evaluation of photolytic pathways should be performed in both
the solid state and in solution. Samples should be photo stressed by exposure to irradiation
significantly beyond the ICH minimum recommended confirmatory levels, both in the ultraviolet
and visible spectral regions.

4.Oxidative Degradation: Oxidative degradation of drug substances in pharmaceutical

formulations is well documented. Although exact mechanistic details about what promotes
reaction between drug substances (RH in the schematic below) and molecular oxygen in
pharmaceutical formulations is not understood fully, such reactions are generally thought to be
in the category of auto-oxidation process.
DRUG PROFILE
Name
Glipizide
Accession Number
DB01067 (APRD00436)
Type
Small Molecule
Groups
Approved, Investigational
Description
An oral hypoglycemic agent which is rapidly absorbed and completely
metabolized.

STRUCTURE

Synonyms
1-cyclohexyl-3-({p-[2-(5-
methylpyrazinecarboxamido)ethyl]phenyl}sulfonyl)urea
Glipizida
Glipizide
Glipizidum
N-{4-[β-(5-methylpyrazine-2-carboxamido)ethyl]benzenesulphonyl}-
N'-cyclohexylurea

International/Other Brands
Aldiab / Digrin / Dipazide / Glibenese (Pfizer) / Glibénèse (Dexo) / Glibetin
(Johnson) / Glican / Glidiab / Glipid / Glipin (Swiss Pharm) / Glix (YSP) / Gluco-
Rite / Glucolip / Glucozide / Glupitel / Glupizide / Glyde / Glydiazinamide /
Melizide / Mindiab (Pfizer) / Minidab / Minidiab (Pfizer) / Minodiab / Napizide /
Ozidia / Sucrazide / Zitrol XR (Square)
Categories
Alimentary Tract and Metabolism
Blood Glucose Lowering Agents
BSEP/ABCB11 Substrates
Cytochrome P-450 CYP2C9 Substrates
Cytochrome P-450 CYP3A Substrates
Cytochrome P-450 CYP3A4 Substrates
Drugs Used in Diabetes
Hypoglycemia-Associated Agents
Oral Hypoglycemics
Sulfones
Sulfonylurea Compounds
Sulfonylureas
Sulfur Compounds
UGT1A1 Substrates
Urea
CAS number
29094-61-9
Weight
Average: 445.535
Monoisotopic: 445.178375067
Chemical Formula
C21H27N5O4S

IUPAC Name
N-[2-(4-{[(cyclohexylcarbamoyl)amino]sulfonyl}phenyl)ethyl]-5-
methylpyrazine-2-carboxamide

Indication
For use as an adjunct to diet for the control of hyperglycemia and its associated
symptomatology in patients with non-insulin-dependent diabetes mellitus
(NIDDM; type II), formerly known as maturity-onset diabetes, after an adequate
trial of dietary therapy has proved unsatisfactory.
Pharmacodynamics
Glipizide, a second-generation sulfonylurea, is used with diet to lower blood
glucose in patients with diabetes mellitus type II. The primary mode of action of
glipizide in experimental animals appears to be the stimulation of insulin
secretion from the beta cells of pancreatic islet tissue and is thus dependent on
functioning beta cells in the pancreatic islets. In humans glipizide appears to
lower the blood glucose acutely by stimulating the release of insulin from the
pancreas, an effect dependent upon functioning beta cells in the pancreatic islets.
In man, stimulation of insulin secretion by glipizide in response to a meal is
undoubtedly of major importance. Fasting insulin levels are not elevated even on
long-term glipizide administration, but the postprandial insulin response continues
to be enhanced after at least 6 months of treatment. Some patients fail to respond
initially, or gradually lose their responsiveness to sulfonylurea drugs, including
glipizide.
Mechanism of action
Sulfonylureas likely bind to ATP-sensitive potassium-channel receptors on the
pancreatic cell surface, reducing potassium conductance and causing
depolarization of the membrane. Depolarization stimulates calcium ion influx
through voltage-sensitive calcium channels, raising intracellular concentrations of
calcium ions, which induces the secretion, or exocytosis, of insulin.

Absorption
Gastrointestinal absorption is uniform, rapid, and essentially complete.
Volume of distribution
11 L
Protein binding
98-99%, primarily to albumin.
Metabolism
Hepatic. The major metabolites of glipizide are products of aromatic
hydroxylation and have no hypoglycemic activity. A minor metabolite which
accounts for less than 2% of a dose, an acetylaminoethyl benzine derivatives, is
reported to have 1/10 to 1/3 as much hypoglycemic activity as the parent
compound.

Route of elimination
The primary metabolites are inactive hydroxylation products and polar conjugates
and are excreted mainly in the urine.
Half life
2-5 hours

Toxicity
The acute oral toxicity was extremely low in all species tested (LD50 greater than
4 g/kg). Overdosage of sulfonylureas including glipizide can produce
hypoglycemia.
Affected organisms
Humans and other mammals

DRUG INTERACTION
(R)-warfarin Glipizide may increase the
anticoagulant activities of (R)-warfarin.
(S)-Warfarin Glipizide may increase the
anticoagulant activities of (S)-Warfarin.
2,4-thiazolidinedione The risk or severity of
hypoglycemia can be increased when Glipizide is combined with 2,4-
thiazolidinedione.
3,5-diiodothyropropionic acid The metabolism of 3,5-
diiodothyropropionic acid can be decreased when combined with Glipizide.
4-hydroxycoumarin The metabolism of 4-
hydroxycoumarin can be decreased when combined with Glipizide.
5-(2-methylpiperazine-1-sulfonyl)isoquinoline The therapeutic efficacy of
Glipizide can be increased when used in combination with 5-(2-methylpiperazine-
1-sulfonyl)isoquinoline.
5-androstenedione The metabolism of 5-
androstenedione can be decreased when combined with Glipizide.
6-Deoxyerythronolide B The metabolism of
Glipizide can be decreased when combined with 6-Deoxyerythronolide B.
6-O-benzylguanine The metabolism of 6-O-
benzylguanine can be decreased when combined with Glipizide.
Abacavir Abacavir may decrease the
excretion rate of Glipizide which could result in a higher serum level.

Food Interactions
Avoid alcohol.
Avoid sugar and sugary food.
Take 30-60 minutes before breakfast.
LITERATURE REVIEW
1.DEVELOPMENT AND VALIDATION OF UV SPECTROPHOTOMETRIC METHOD
FOR ESTIMATION OF GLIPIZIDE IN BULK AND PHARMACEUTICAL DOSAGE
FORMS

Vaishali V. Karkhanis*, Anandkumari D. Captain and Priti H. Patel

A.R. College of Pharmacy and G.H. Patel Institute of Pharmacy Vallabh Vidhyanagar, Dist: Anand-
388120, Gujarat, India

A selective, simple, accurate and reproducible spectrophotometric method has been developed for the
estimation of Glipizide in bulk and pharmaceutical formulation. Glipizide is a second generation
sulfonylurea which lowers blood glucose in patients with diabetes mellitus type II. The drug obeyed the
Beer’s law and showed good correlation. It showed absorption maxima at 276 nm in 0.1N NaOH. The
developed method was validated with respect to linearity, accuracy and precision in accordance with the
requirements of ICH guidelines. The linearity was observed between 10-30μg/ml having line equation
Y=0.0283X - 0.0248 with correlation coefficient of 0.999.The limit of quantification and limit of detection
were found to be 1.643 and 0.542ug/ml respectively. Moreover, the proposed analytical method is thus
potentially useful for a routine laboratory because of its simplicity, rapidity, precision and accuracy.

2. Determination of Glipizide, Glibenlamide and Glimeperide in a Tablet Dosage Form in

the Presence of Metformin Hydrochloride by Ion Pair –Reversed Phase Liquid
Chromatographic Technique

Anna Pratima G etal

Reasearch gate 1(2):doi:10.4172/2155-9872.1000105 · November 2010

The present work describes the development and validation of an isocratic HPLC method for the stability
indicating assay of Glipizide (GPZ), Glibenclamide (GBD) and Glimeperide (GMD) in the presence of
Metformin hydrochloride (MET) in pharmaceutical dosage forms using ion pair-reversed phase liquid
chromatographic Technique. The ion pairing agent used was tetrabutyl ammonium hydrogen sulphate
(TBHS). The TBHS 0.030 molar solution in water with pH 6.0 used as buffer. The composition of buffer
with acetonitrile used was 50:50 (v/v) on reversed phase column bonded with octadecyl silane. The wave
length used was 225 nm. The resolution between the closest peaks Glimeperide and Glibenclamide was
more than 1.5 and all the three drugs gives a linear response (r2>0.999). The method used for all the three
substances were found selective, precise, accurate and robust. The method can be used for quality control
assay of the bulk and in fi nished dosage form as single component and combine with Metformin
hydrochloride. The purpose of the method to study individually the stability of Glipizide, Glibenclamide
and Glimeperide in the presence of Metformin hydrochloride as any such method is not reported so far.

3.Development and validation of RP-HPLC-UV method for the determination of Glipizide

in human plasma

M Atif etal
US National Library Of Medicine . 2013 Mar; 5(1): 26–29.
A simple, sensitive and selective HPLC method with UV detection for determination of Glipizide in
human plasma was developed. Liquid–liquid extraction method was used to extract the drug from the
plasma samples. Chromatographic separation of Glipizide was achieved using C18 column (ZORBAX
ODS 4.6 × 150 mm). The mobile phase was comprised of 0.01 M potassium dihydrogen phosphate and
acetonitrile (65:35, v/v) adjusted to pH 4.25 with glacial acetic acid. The analysis was run at a flow rate
of 1.5 mL/min with an injection volume was 20 μL. The detector was operated at 275 nm. The calibration
curve was linear over a concentration range of 50–1600 ng/mL. Intra-day and inter-day precision and
accuracy values were below 15%. The limit of quantification was 50 ng/mL and the mean recovery was
above 98%. Freeze-thaw, short-term, long-term and post-preparative stability studies showed that
Glipizide in plasma sample was stable. The method may be successfully applied to analyze the Glipizide
concentration in plasma samples for bioavailability and bioequivalence studies.
4. Quality by Design Approach for the Development and Validation of Glipizide, an
Antidiabetic Drug, by RP-UPLC with Application to Formulated Forms and Urine
Cizo M.Xavier etal
ISRN Chromatography
Volume 2013, Article ID 738397

Quality by design (QbD) refers to the achievement of certain predictable quality with desired and
predetermined specifications. The objective of this study was to develop and demonstrate an integrated
multivariate approach to develop and quantify the constituent concentrations of glipizide (GPZ) drug in
its pure and tablet forms. The method was developed using Zorbax Extend C-18 (50 mm × 4.6 mm ×
1.8 μm) column with mobile phase consisting of a mixture of phosphate buffer of pH 3.5 and acetonitrile
(60 : 40 v/v). The method fulfilled validation criteria and was shown to be sensitive, with limits of
detection (LOD) and quantitation (LOQ) of 0.001 and 0.005 μg mL−1, respectively. The percentage
relative standard deviations for robustness and ruggedness were observed within the range of 0.1 and 0.99.
The calibration graph was linear in the range of 0.005–300 μg mL−1. The applicability of the method was
shown by the analysis of formulated drug and spiked urine samples. The proposed method can be used
for routine analysis in quality control laboratories for its bulk and formulated product, and this is the first
UPLC method reported for the assay of GPZ in bulk, formulated form and urine.

5. A Rapid and Simple UPLC–MS-MS Method for Determination of Glipizide in Human

Plasma and Its Application to Bioequivalence Study
Xiangjun Qiu etal
Journal of Chromatographic Science, Volume 53, Issue 1, 1 January 2015,
In this study, a simple, rapid and sensitive ultra performance liquid chromatography–tandem mass
spectrometry method is described for the determination of glipizide in human plasma samples using
carbamazepine as the internal standard (IS) from bioequivalence assays. Sample preparation was
accomplished through protein precipitation with methanol, and chromatographic separation was
performed on an Acquity BEH C18 column (2.1 mm × 50 mm, 1.7 μm) with gradient profile at a flow
rate of 0.4 mL/min. Mass spectrometric analysis was performed using an QTrap5500 mass spectrometer
coupled with an electrospray ionization source in the positive ion mode. The multiple reaction monitoring
transitions of m/z 446.1 → 321.0 and m/z 237.1 → 194.2 were used to quantify for glipizide and IS. The
linearity of this method was found to be within the concentration range of 10–1,500 ng/mL for glipizide
in human plasma. Only 1.0 min was needed for an analytical run. The method was applied to a
bioequivalence study of two drug products containing glipizide in human plasma samples.
6. Spectrophotometric Methods for the Determination of Anti-diabetic Drug Glipizide in
Pure and Pharmaceutical Formulations
Nasrin Banu Shaikh Ismail
Mangalore University, INDIA Received 9 March 2016 ▪ Revised 4 May 2016 ▪ Accepted 5 May 2016
Four simple and precise spectrophotometric methods were developed and optimized for the assay of
glipizide (GPZ) in pure form and in its pharmaceutical preparations. Method A was based on the reaction
of drug with 1,2-naphthoquinone-4-sulfonate (NQS) in alkaline medium to form orange colored product
(λmax 456 nm). In the method B, drug reduced the reagent tetrazolium blue (TB) which lead to the
formation of intense violet colored formazan (λmax 515 nm). The method C employed Folin-Ciocalteu
reagent (FC) that reacted with drug in alkaline medium to form blue colored complex (λmax 760 nm).
Method D was based on the reduction of iron(III) to iron(II) by the drug and their complexation with
bathophenanthroline (BPT) to form pink colored complex (λmax 547 nm). Linearity of Beer’s plots was
observed in the concentration range of 10.00 – 100.00 µg mL-1 , 2.00 – 22.00 µg mL-1 , 5.00 – 40.00 µg
mL-1 , 0.50 – 7.50 µg mL-1 for method A, B, C and D respectively. The proposed methods were validated
and values of analytical parameters like molar absorptivity, Sandell’s sensitivity, limits of detection and
quantification were also calculated. The methods were found to be selective for the quantitative
determination of GPZ in commercially available formulations.
7.Development and validation of RP-HPLC method for quantification of glipizide in
biological macromolecules
Nihar Ranjan Pani etal International Journal Of Biological Macromolecules April 2014, Pages 65-71

Glipizide (GPZ) has been widely used in the treatment of type-2 diabetics as insulin secretogague.
Multiunit chitosan based GPZ floating microspheres was prepared by ionotropic gelation method for
gastroretentive delivery using sodiumtripolyphosphate as cross-linking agent. Pharmacokinetic study of
microspheres was done in rabbit and plasma samples were analyzed by a newly developed and validated
high-performance liquid chromatographic method. Method was developed on Hypersil ODS-18 column
using a mobile phase of 10 mM phosphate buffer (pH, 3.5) and methanol (25:75, v/v). Elute was monitored
at 230 nm with a flow rate of 1 mL/min. Calibration curve was linear over the concentration range of
25.38–2046.45 ng/mL. Retention times of GPZ and internal standard (gliclazide) were 7.32 and 9.02 min
respectively. Maximum plasma drug concentration, area under the plasma drug concentration–time curve
and elimination half life for GPZ floating microspheres were 2.88 ± 0.29 μg mL−1,
38.46 ± 2.26 μg h mL−1 and 13.55 ± 1.36 h respectively. When the fraction of drug dissolved from
microspheres in pH 7.4 was plotted against the fraction of drug absorbed, a linear correlation (R2 0.991)
was obtained in in vitro and in vivo correlation study.
8. THE COMPLITE REVIEW ON ANALYTICAL AND FORMULATION
TECHNIQUES OF GLIPIZIDE

Rashmi Balkate etal Research Gate January 2018

Glipizide is second generation Short acting sulfonylurea prescribed for treatment of type II diabetes
Mellitus. It is with short biological half-life of 3.4 ± 0.7 hrs and metabolized in the liver and excreted in
the urine largely as inactive metabolites. The clinical and pharmaceutical analysis of drug requires
analytical procedures along with pharmacokinetic and pharmacodynamic data with stability study for any
analysis of drug. In the present review we have compiled different published analytical methods for
determination of glipizide in pharmaceutical methods. The table no 1 indicate Analytical method
development and validation of single Glipizide drug by HPLC method; while table no 2 indicates
Analytical method development and validation of glipizide with combination of other drugs by HPLC
method. In the literature review table no 3 and 4 indicates Analytical method development and validation
of glipizide as well as other drugs in combined forms by UV spectrophotometer. This literature review
also involved tabulated information about various formulations available of glipizide along with their
method of formulation and polymers used in the formulations.

9. Rapid and Reliable Determination of Glipizide in Pharmaceutical Samples by HPLC and

Its Degradation Study

Rajendraprasad etal

Austin J Anal Pharm Chem. 2017; 4(1): 1080.

A simple, specific and rapid high-performance liquid chromatographic (HPLC) method for the
determination of glipizide (GPZ) in pharmaceutical sample is described. Reversed phase chromatography
was performed on an Inertsil ODS 3V (150 × 4.6mm; 5μm particle size) column with an isocratic mobile
phase consisting of 10mM potassium dihydrogen phosphate (pH 3.9) and methanol (60:40 v/v). The
effluent was monitored on a uv detector at 220nm and the column temperature was maintained at 35°C.
Linear response (r=0.9999) was observed over the range, 1-450μgmL-1; and the limits of detection (LOD)
and quantification (LOQ) were calculated to be 0.03 and 0.09 μgmL-1, respectively. Both intra-day and
inter-day precisions at three tested concentrations were excellent, with %RSD values of <1% and the
accuracy was better than 1.5% (RE). The method was also validated for robustness, ruggedness and
selectivity and the results were satisfactory. The method was applied to the determination of GPZ in tablets
and the results agreed well with the label claim and those obtained by European Pharmacopeial method.
Entire assay was complete in less than 10min. As part of degradation study, the drug was subjected to
acid-, base-, peroxide-, heat-, and light-induced stress conditions, and the drug was found to be susceptible
to degradation under oxidation, and inert to other conditions.

10.SIMULTANEOUS ESTIMATION OF METFORMIN AND GLIPIZIDE BY RP-HPLC

AND ITS VALIDATION

Sri Lakshmi D*, Jane T Jacob, Srinivas D, Satyanarayana D

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES SJIF Impact Factor

5.210 Volume 4, Issue 09, 740-750

A simple, accurate, economical and precise reverse phase high performance liquid chromatographic (RP-
HPLC) method has been developed for the simultaneous determination of Metformin and Glipizide. The
separation was achieved on Intersil C 18 column (250 x 4.6 mm, 5 µm) as stationary phase with a mobile
phase comprising of Phosphate buffer p H (8.0):Acetonitrile (50:50) in an isocratic mode, at a flow rate
of 2 ml/min. The detection was monitored at 257 nm. The retention time of Metformin and Glipizide were
2.41 min and 4.21 min respectively. The linearity was found to be in the range of 60-140 µg/ml and 3.6-
8.4 µg/ml for Metformin and Glipizide respectively with correlation coefficient of 0.999. The proposed
method was validated according to ICH guidelines for parameters like linearity, accuracy, precision and
specificity. All validation parameters were within the acceptable range. The developed method was
successfully applied for the estimation of Metformin and Glipizide in pure and pharmaceutical dosage
form.

11.METHOD FOR ESTIMATION OF GLIPIZIDE IN BULK AND PHARMACEUTICAL

DOSAGE FORMS

Vaishali V. Karkhanis*, Anandkumari D. Captain and Priti H. Patel

A.R. College of Pharmacy and G.H. Patel Institute of Pharmacy Vallabh

Vidhyanagar, Dist: Anand-388120, Gujarat, India

Vol-2 suppl 2014

A selective, simple, accurate and reproducible spectrophotometric method has been

developed for the estimation of Glipizide in bulk and pharmaceutical formulation.
Glipizide is a second generation sulfonylurea which lowers blood glucose in
patients with diabetes mellitus type II. The drug obeyed the Beer’s law and
showed good correlation. It showed absorption maxima at 276 nm in 0.1N NaOH.
The developed method was validated with respect to linearity, accuracy and
precision in accordance with the requirements of ICH guidelines. The linearity was
observed between 10-30μg/ml having line equation Y=0.0283X - 0.0248 with
correlation coefficient of 0.999.The limit of quantification and limit of detection
were found to be 1.643 and 0.542ug/ml respectively. Moreover, the proposed
analytical method is thus potentially useful for a routine laboratory because of its
simplicity, rapidity, precision and accuracy.

12.Development and Validation of UV Spectrophotometric Method for Simultaneous

Estimation of Metformin and Glipizide in Tablet Dosage Form

K. Ganesh, G. Nikitha, D. Sireesha, B. Vasudha

Article ·May 2016
A simple, rapid and precise spectrophotometric method has been developed for
simultaneous estimation of Metformin and Glipizide. The method involved
estimation of Metformin and Glipizide by simultaneous equation at 272nm and
232nm respectively in their solution in water. This method was validated with
respect to linearity, accuracy, precision, LOD and LOQ. Beer’s law obeyed in the
concentration range of 5-25μg/ml and 20-50μg/ml for Metformin and Glipizide
respectively with the correlation coefficient of above 0.99. Limit of detection and
quantification values were determined to be 0.214μg/ml and 0.649μg/ml for
Metformin and 0.608μg/ml and 1.854μg/ml for Glipizide respectively. Mean
recovery of Metformin and Glipizide were found to be in the range of 98-102%
signifies the accuracy of the method. The method was found to be precise as %RSD
was less than 2.
13. Ultraviolet Spectrophotometric Method for Determination of Glipizide in Bulk and
Tablet Dosage Formulation

Nipanikar Madhuri1, Shrikrishna Baokar1*, Undare Santosh2, Patil R. N.

Vol-3 suppl-2016

Glipizide (GPZ) is chemically 1cyclohexyl3

[[p[2(5methylpyrazinecarboxamido)ethyl]phenyl] sulfonyl]urea, used in the
treatment of type II diabetes mellitus. The drug is commercially available as tablets
for oral administration. In the present work three simple, economical, precise and
accurate UV spectrophotometrically methods have been developed for the
estimation of GPZ in bulk and pharmaceutical formulation. The present method has
been developed in distil 0.1 N NaOH which makes it economic and reproducible.
An absorption maximum was obtained at 227 nm. The method is validated using
ICH Q2R1 guideline for various parameters like precision, accuracy, etc. Drug
follows linearity in concentration range 1-10 μg/ml with correlation coefficient
value (r2) 0.999. The accuracy study was performed by recovery checking at three
different concentrations i.e., 80%, 100% and 120 %. The % recovery was found
well in limit. The precision of the method was studied as an intra-day (% R.S.D. =
0.44 %) and for inter-day (% R.S.D. = 0.22 %) variations. The % R.S.D. value
less than 2 indicate that the method is precise. The proposed method is a cost-
effective quality-control tool for routine analysis of GPZ in pharmaceutical dosage
form.

14. Development of validated UV spectrophotometric method for the simultaneous

estimation of metformin hydrochloride and glipizide in tablet dosage form

Manoj S. Charde

Vol-2 suppl 2005

A simple precise reproducible U.V. Spectrophotometric method have been

developed and validated for the simultaneous estimation of MET and GPZ in tablet
dosage form. This paper describes the simultaneous equation method as a
quantification parameter which involves the measurement of absorbance of MET
and GPZ at 237 nm and 234.0 nm respectively. MET and GPZ both obeyed linearity
in the range of 2?g/mL to 20 ?g/mL. The recovery studies shows %RSD for MET
0.005, 0.12, 0.65 and for GPZ 0.008, 0.54,0.25 by SEM method. The results of
analysis have been validated statistically for accuracy, Precision, Repeatability, and
Ruggedness. The method was successfully applied to the determination of these
drugs in pharmaceutical dosage form
15. Ultraviolet Spectrophotometric Method for Determination of Glipizide in Presence of
Liposomal/Proliposomal Turbidity
Neelkant Prasad, Roshan Issarani, and Badri Prakash Nagori
Journal of Spectroscopy
Volume 2013,
A simple and sensitive ultraviolet spectrophotometric method for quantitative
estimation of glipizide in presence of lipid turbidity is described to avoid false
estimation due to diffraction by turbidity. UV detection was performed at 230 nm,
225 nm, and 235 nm, and the calibration curve was plotted between resultant of
absorbance of [230 nm − (225 nm + 235 nm)/2] and concentration of analyte. The
calibration curve was linear over the concentration range tested (1–20 μg/mL) with
limit of detection of 0.27 μg/mL and limit of quantification of 0.82 μg/mL. Percent
relative standard deviations and percent relative mean error, representing precision
and accuracy, respectively, for clear as well as turbid solutions, were found to be
within acceptable limits, that is, always less than 0.69 and 0.41, respectively, for
clear solution and 0.65 and 0.47, respectively, for turbid solution. Conclusively, our
method was successfully applied for the determination of glipizide in clear as well
as turbid solutions, and it was found that the drug analyte in both types of solutions
can be detected from the same calibration curve accurately and precisely and
glipizide entrapped in the liposomes or in proliposomal matrix was not detected.

16. SPECTROPHOTOMETRIC DETERMINATION OF GLIPIZIDE IN BULK AND

TABLET DOSAGE FORM BY ABSORPTION MAXIMA, FIRST ORDER
DERIVATIVE SPECTROSCOPY AND AREA UNDER THE CURVE

DINESH R. RATHOD, MANJUSHA N. DOLE*, SANJAY D. SAWANT

Asian Journal of Pharmaceutical and Clinical Research

Vol 5 Suppl 3 2012

Glipizide (GZ) is chemically

1cyclohexyl3[[p[2(5methylpyrazinecarboxamido)ethyl]phenyl] sulfonyl]urea,
used in the treatment of type II diabetes mellitus. The drug is commercially
available as tablets for oral administration. In the present work three simple,
economical , precise and accurate UV spectrophotometric methods have been
developed for the estimation of glipizide in bulk and pharmaceutical formulation.
Method A is absorption maxima method in which λmax was found to be 274 nm.
Method B is first order derivative spectroscopy where drug showed
λ maxima=286 nm and λ minima=263nm. Amplitude difference (dA/dλ) was
calculated and was plotted against concentration and regression equation was
calculated. Method C is area under the curve (AUC) in which area in the
wavelength range of 255 nm -295 nm was selected for analysis of glipizide.
Linearity was observed in the concentration range 5- 25 μg/ml ( r 2 =0.999) for all
the three methods. The % assay for the marketed formulation for absorption
maxima, first order derivative and area under the curve method was found to be
99.03%, 10 0.16% and 99.06% respectively. The methods were validated with
respect to linearity, precision and accuracy studies. Recovery studies for absorption
maxima, first order derivative and area under the curve was found to be 100.83
%, 99.41% and 100.51% respectively. The methods were found to
be simple, precise and accurate and can be employed for routine quality control
analysis of glipizide in bulk as well as from its dosage
form.

17. Devlopement and Validation of UV-Visible Spectrophotometric Method for

Simultaneous Determination of pioglitazone Hydrochloride, Metformin Hydrochloride and
Glipizide in its Bulk and Pharmaceutical Dosage Form (Tablet)

L Adhikari

International Journal of ChemTech Research CODEN( USA): IJCRGG ISSN : 0974-4290 Vol.4, No.2,
pp 625-630, April-June 2012

A simple ,precise, rapid and selective spectroscopic method is used to Quantify

three antidiabetics in multicomponent formulations in the present study.Three wave
length spectroscopic method and Multiwavelength method was carried out for
determination of Metformin Hydrochloride,Glipizide,Pioglitazone Hydrochloride
in their bulk and preparations using Acetonitrile: Methanol: Water in the proportion
of 5:4:1.The λmax was found at 236.5nm, 226.4nm and 227.3nm respectively.
The isobestic point was found to be 254 nm. Method:II is based on multiwavelength
spectroscopy. All the three drugs obeys the Beer-Lambert limit within the
concentration range of
5-50μg/ml.%COV<2 and S.D<2. Assay results from both the methods with
different formulations shows purity around 99.95% to 102.10%.Validation study
reveals that the methods are specific, accurate and precise. High recovery and low
%COV reveals the reliability and good acceptance of the quantitative study in
formulations. The method can be used for routine quantitative analysis of
metformin,glipizide and pioglitazone in pure and tablet
dosage forms.

18. Spectrophotometric Methods for the Determination of Anti-diabetic Drug Glipizide in

Pure and Pharmaceutical Formulations
Nasrin Banu Sheikh
Eurasian Journal of Analytical Chemistry
Vol 5 suppl 4 2015
Four simple and precise spectrophotometric methods were developed and
optimized for the assay of glipizide (GPZ) in pure form and in its pharmaceutical
preparations. Method A was based on the reaction of drug with 1,2-
naphthoquinone-4-sulfonate (NQS) in alkaline medium to form orange colored
product (λmax 456 nm). In the method B, drug reduced the reagent tetrazolium blue
(TB) which lead to the formation of intense violet colored formazan (λmax 515 nm).
The method C employed Folin-Ciocalteu reagent (FC) that reacted with drug in
alkaline medium to form blue colored complex (λmax 760 nm). Method D was based
on the reduction of iron(III) to iron(II) by the drug and their complexation with
bathophenanthroline (BPT) to form pink colored complex (λmax 547 nm). Linearity
of Beer’s plots was observed in the concentration range of 10.00 – 100.00 µg mL-
1
, 2.00 – 22.00 µg mL-1, 5.00 – 40.00 µg mL-1, 0.50 – 7.50 µg mL-1 for method A,
B, C and D respectively. The proposed methods were validated and values of
analytical parameters like molar absorptivity, Sandell’s sensitivity, limits of
detection and quantification were also calculated. The methods were found to be
selective for the quantitative determination of GPZ in commercially available
formulations.

19. DEVELOPMENT AND VALIDATION OF SIMULTANEOUS EQUATION

METHOD FOR BILAYER FLOATING TABLET OF GLIPIZIDE AND LISINOPRIL

Sheetal Buddhadev

International Journal of Innovative Pharmaceutical Sciences and Research.

Vol 3 suppl 2 2016
To develop and validate a simple& accurate Spectrophotometry method for
simultaneous ,equation method of Glipizide and Lisinopril in their combined
tablets.The method was based on Simultaneous Equation method. Here 204.5 nm
was selected for the estimation of Lisinopril &
224.5 nm selected for estimation of Glipizide. Glipizide and Lisinopril showed
same linearity in the range of 0.5-15μg/ml. Then the method was validated by
validation parameters and it show result where lie within its acceptance criteria as
per ICH Q2 (R1) guideline. Hence, it can be successfully used for the routine
analysis of Glipizide and Lisinopril in their combined pharmaceutical dosage forms

20. Development and Validation of Novel UV-Spectrophotometric Method for

the Estimation of Flurbiprofen and Glipizide Using Hydrotropic
Solubilization Technique in Bulk and Pharmaceutical Dosage Form
Srujani Ch1*, Tanuja A1, Varanasi S N Murthy1

American Journal Of Pharmatech Research

Vol 1 2017

Effective and advantageous Hydrotropic Solubilization technique has been

developed for the estimation of two drugs i.e, Flurbiprofen and Glipizide in bulk
and its pharmaceutical formulations. Hydrotropic Solubilization technique is one
of the aqueous solubility enhancing methods employed for the poorly water
soluble drugs and found to be simple, precise and cost effective. Solvents like
Piperazine, Urea, Sodium Salicylate, Sodium benzoate etc are the commonly used
as hydrotropic solventsin different concentrations. The use of these hydrotropic
solvents are of proper choice since the use of organic solvents can be reduced as it
is hazardous, costlier and causes environmental pollution. In this context, 1M
piperazine has been used as a solubilizing agent to enhance solubility of both the
drugs, Flurbiprofen and Glipizide. The absorption maximum of Flurbiprofen and
Glipizide was found to be at 246nm and276nm in Zero order derivative spectrum
(Method A), calculation of Area Under Curve (AUC)(Method B) was done in the
wavelength range of 236-256nm for Flurbiprofen and 266-286nm for Glipizide.
The Beer-Lambert’s law has been followed in the concentration range of 2-
10µg/ml for Flurbiprofen and 5-35µg/ml for Glipizide for both the methods. The
methods were validated as per ICH guidelines and all the validation parameters
were found to be within the acceptable range. The developed methods were
successfully practiced to estimate the amount of Flurbiprofen and Glipizide in bulk
and pharmaceutical dosage forms in routine analysis.

21. UV SPECTROPHOTOMETRIC METHOD DEVELOPMENT AND

QUANTITATIVE ESTIMATION OF GLIPIZIDE IN BULK AND
PHARMACEUTICAL DOSAGE FORMS
Internatinal journal of drug research and technology
Anu Vijan
Vol 2009
The present study demonstrate a simple, precise and accurate UV spectrophotometric
method development for the quantitative estimation of glipizide in bulk and
pharmaceutical dosage forms. The analysis was carried out by using Elico SL 159
UV –Visible spectrophotometer with 1cm matched quartz cells. In this study, the
zero order spectrum of glipizide in the presence of phosphate buffer (pH 3.8) is
conducted and measured the absorbance at λmax 230 nm. The method was verified
by using various validation parameters such as linearity, accuracy, precision. The
calibration curve was linear over the concentration range tested (1–50 μg/mL). The
accuracy and precision studies were carried out which is less than 2% that indicates
good accuracy and precision values. There is no interference of excipients used in
the formulation as low % RSD values in the recovery studies. These results
indicate that the method shows a practical application as a quality control tool for
analysis of the drug in its tablet dosage forms in pharmaceutical industries. The
developed method was validated according to ICH-Q1C guidelines and it is
applicable for the analysis of bulk drug in its tablet dosage forms.