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PREFORMULATION &
ANALYTICAL METHOD DEVELOPMENT &
Validation
Chapter 2: Preformulation Studies
47
2.1. Preformulation and Standardization of Drugs
Preformulation is an exploratory activity that begins early in drug development.
Preformulation studies are designed to determine the compatibility of excipients with the
active substance for a biopharmaceutical, physicochemical, and analytical investigation
in support of promising experimental formulations. These studies are conducted to form
the basis for the rational of formulation design. Data from preformulation studies provide
the necessary groundwork for formulation attempts.
The selected drugs and excipients were standardized as per respective pharmacopoeial
specifications, or as per the manufacturers’ specifications. The Certificates of Analysis
provided by the suppliers in case of gift samples have been duly included in the
appropriate sections.
2.1.1. Mepivacaine HCl (MH)
Chemical Name: - (±)-1-Methyl-2′, 6′ pipecoloxylidide monohydrochloride
Molecular Formula: - C15H22N2O. HCl
Molecular Weight: - 282.81
Appearance: A white crystalline powder
Solubility: Freely soluble in water and methanol, very slightly soluble in methylene
chloride.
Melting point: 255 - 2620C with partial decomposition
2.1.2. Mepivacaine Base (MB)
Chemical Name: - N-(2, 6-dimethylphenyl)-1-methylpiperidine-2-carboxamide
Molecular Formula: - C15H22N2O
Molecular weight: - 246
Appearance: Odourless, white powder
Chapter 2: Preformulation Studies
48
Solubility: Water solubility is 7000 mg/L. It is soluble in alcohol, methanol, chloroform,
acetone and phosphate buffer pH 6.8.
Melting point: 149-153°C
2.1.3. Lidocaine HCl
Chemical Name:- 2',6'-Acetoxylidide, 2-(diethylamino)-, hydrochloride
Molecular Formula: - C14H22N2O . HCl
Molecular weight: - 270.8
Appearance: White crystalline solid powder
Solubility: Very soluble in water and alcohol, soluble in chloroform, insoluble in ether
Melting point: 77°C
Dissociation constant: pKa is found to be 13.78
Indication: For production of local or regional anesthesia by infiltration techniques such
as percutaneous injection and intravenous regional anesthesia by peripheral nerve block
techniques such as brachial plexus and intercostal and by central neural techniques such
as lumbar and caudal epidural blocks.
Lidocaine HCl was used as a reference drug for comparative investigations on developed
formulations. Hence was procured, standardized and analytical method was developed.
2.2. Standardization of Drugs and Excipients
2.2.1. Monographic Evaluation of Mepivacaine HCl, Mepivacaine Base and
Lidocaine HCl
Mepivacaine base and its hydrochloride salt were procured from Hagzhou
Verychem Science and Technology Co. Ltd. China. Both the drug forms were
standardized as per USP monograph; purity and identity were checked with Certificates
of Analyses provided by supplier. Lidocaine HCl was procured as a gift sample from
Gufic Biosciences Limited, Mumbai. It was standardized as per monograph and purity
and identity were checked with Certificate of Analysis provided by supplier.
Mepivacaine HCl, Mepivacaine Base and Lidocaine HCl were tested for the following-
Chapter 2: Preformulation Studies
49
Appearance: Colour of drugs was observed visually.
Solubility: Solubility was checked in alcohol, methanol, chloroform, acetone and
phosphate buffers of different pH.
Identification tests: Infrared spectrum of drugs was investigated using FTIR Infrared
Spectrophotometer using potassium disk method. Spectrum was scanned over the
wave number range 4000-400 cm-1
.
Loss on drying: Drug (1gm) was weighed and dried in an oven at 100°C- 105°C to
constant weight for 4 hours. The weight was again recorded.
Melting point: This was determined using melting point testing apparatus.
Assay: Percent drug content was considered as mentioned in Certificate of Analysis
of drug obtained from the suppliers and confirmed by the analytical method described
in later section.
The results of the standardization tests are mentioned in Tables 2.1-2.5 & Figs 2.1-2.4.
Table 2.1: Monographic evaluation of Mepivacaine HCl
TESTS SPECIFICATIONS RESULTS
Appearance White or almost white powder Complied
Identification By IR Complied
Loss on drying (w/w) 1.0% max 0.1%
Melting point 260-2620C 262°C
Assay 98.5-101.0% 99.97%
Table 2.2: Monographic evaluation of Mepivacaine Base
TESTS SPECIFICATIONS RESULTS
Appearance White or almost white powder Complied
Identification By IR Complied
Loss on drying (w/w) 1.0% max 0.1%
Melting point 149–153°C 152°C
Assay 98.0% -102.0 % (On anhydrous basis) 99.87%
Chapter 2: Preformulation Studies
50
Fig 2.1: Certificate of Analysis of Mepivacaine Base & HCl
Fig 2.2: IR spectrum of Mepivacaine HCl and Base
Table 2.3: I.R Frequencies for Mepivacaine HCl and Base
I.R. Frequencies obtained for
Mepivacaine HCl
Frequencies obtained for
Mepivacaine Base
Frequency [cm-1
] Assignment Frequency (cm-1
) Interpretation
2957.14 N-H stretch ~3000 N-H stretching
2488.40 C-H stretch ~2500 C-H stretching
1674.36 HN-C=O (amide) ~1650 HN-C=O(amide)
1537.40 C=C (aromatic) ~1550 C=C(aromatic)
Chapter 2: Preformulation Studies
51
Table 2.4: Monographic evaluation of Lidocaine HCl
Tests Specifications Results
Appearance White or almost white crystalline powder Complied
Solubility Very soluble in water, freely soluble in chloroform and
in ethanol 95%. Practically insoluble in ether
Complied
Identification By IR, To match with working standard Complied
Assay 98.0% -102.0 % ( On anhydrous basis) 99.8%
pH 4-6 4.5
Clarity and
colour of
solution
A 5 %W/V solution in CO2 free water is clear Complied
Fig 2.3: IR spectrum of Lidocaine HCl
Table 2.5: Frequencies obtained for Lidocaine Base
Frequency (cm-1
) Interpretation
3248 cm-1
N-H stretch (sec amine)
1654 cm-1
C=O stretch
1505 cm-1
C=C (aromatic)
Chapter 2: Preformulation Studies
52
Fig 2.4: Certificate of Analysis of Lidocaine HCl
2.2.2. Standardization of Excipients
The choice of the excipients depends on several factors; namely, the drug used, the
process involved, the formulator and the cost of excipients. All the excipients used in the
formulation development were procured from authentic vendors and Certificates of
Analysis were obtained for the same. Some of the important tests were performed as per
monographs and Certificate of Analysis to confirm the quality of the excipients.
Majority of the excipients used for the formulation development work are as listed in
Table 2.6:
Chapter 2: Preformulation Studies
53
Table 2.6: List of Excipients used in Formulation Development
Name of the Excipient Source Use
Ethanol 95% S. D Fine Chemicals, Mumbai. Co-solvent
Isopropyl alcohol S. D Fine Chemicals, Mumbai. Co-solvent, cosurfactant
Propylene glycol S. D Fine Chemicals, Mumbai. Co-solvent
Acetone S. D Fine Chemicals, Mumbai. Solvent
Triethanolamine S. D Fine Chemicals, Mumbai. Neutralizer
Hydroxypropyl
methylcellulose (HPMC)
Aqualon, Mumbai Gelling agent, polymer
Carbopol 980 NF Lubrizol, Mumbai Gelling agent, polymer
Carbopol 971P NF Lubrizol, Mumbai Gelling agent, polymer
Carbopol 974P NF Lubrizol, Mumbai Gelling agent, polymer
Transcutol P Gattefosse Ltd, Mumbai Co-surfactant
Labrasol Gattefosse Ltd, Mumbai Surfactant
Tween 80 S. D Fine Chemicals, Mumbai. Surfactant
Butylated hydroxylanisole S. D Fine Chemicals, Mumbai. Anti-oxidant
Butylated hydroxyltoluene S. D Fine Chemicals, Mumbai. Anti-oxidant
Sodium metabisulphite S. D Fine Chemicals, Mumbai. Anti-oxidant
Poloxamer F68 BASF, Mumbai Film forming polymer
Kollidon® 30 BASF, Mumbai Film forming polymer
Eudragit RL 100 Evonik Degussa, Mumbai Film forming polymer
Oleic acid S. D Fine Chemicals, Mumbai. Penetration enhancer &
oil
Menthol Sigma Aldrich Penetration enhancer
Eugenol Sigma Aldrich Penetration enhancer
Propyl paraben S. D Fine Chemicals, Mumbai. preservative
Methyl paraben S. D Fine Chemicals, Mumbai. preservative
1. Ethanol 95%
It is primarily used as a solvent and as penetration enhancer.
Table 2.7: Monographic evaluation of Ethanol 95%
Tests Specifications Results
Description
Clear, colorless and volatile liquid with
slight characteristic odor and burning taste
Complied
Non-volatile residue Not more than 1.0 mg 0.3 mg
Specific gravity 0.812-0.816 g/mL 0.814
g/mL
Chapter 2: Preformulation Studies
54
2. Isopropyl alcohol (Propane-2-ol)
It is used in cosmetics and pharmaceutical formulations primarily as a solvent in
topical formulations.
Table 2.8: Monographic evaluation of isopropyl alcohol
Tests Observations Specifications and
remarks
Characteristics Clear colourless liquid Complied
Solubility Miscible with benzene, chloroform,
ethanol, ether, glycerine and water.
Soluble in acetone
Complied
Boiling point 81.2 oC Confirmed (82.4
oC)
Specific gravity 0.788 Confirmed (0.786)
3. Propylene glycol
It is widely used as water miscible co-solvent, and penetration enhancer in topical
formulations.
Table 2.9: Monographic evaluation of Propylene glycol
Tests Specifications Results
Description Clear, colorless, viscous, practically
odorless liquid with a sweet, slightly
acrid taste resembling that of glycerin.
Complied
Residue on ignition ≤3.5 mg 2.9 mg
Specific gravity 1.035–1.037 g/mL 1.035 g/mL
4. Hydroxypropyl methylcellulose (HPMC) USP
Hydroxypropyl methylcellulose (HPMC) is partially o-methylated and O-(2
hydroxypropylated) cellulose. HPMC is an odourless and tasteless, creamy-white
coloured fibrous or granular powder. It is available in various grades that vary in
viscosity and extent of substitution. It is used as a suspending and thickening agent in
topical formulations. It has found major application as an emulsifier, suspending agent,
and stabilizing agent in topical gels and ointments.
Chapter 2: Preformulation Studies
55
Table 2.10: Standardization of HPMC K4M USP XXIV
Test Observation Specification and
Inference
Characteristics Odourless, tasteless, white granular
powder.
Complied
Solubility Soluble in cold water forming a viscous
solution. Insoluble in alcohol, ether,
chloroform.
Complied
Apparent viscosity
cPs (USP)
4000 Passes
Loss on drying (%) 2.5 Passes (NMT 5%)
Sieve analysis (%)
% Through#40
% Through#100
99.8
94
Min 99
Min 90
pH, in water (1%) 7 Passes (5-8)
5. Tween 80 (Polysorbate 80) USP
Tween 80 is a non ionic surfactant and emulsifier derived from polyethoxylated sorbitan
and oleic acid. It is viscous, water soluble yellow liquid. The hydrophilic groups in this
compound are polyethers also known as polyoxyethylene groups which are polymers of
ethylene oxide.
Table 2.11: Standardization of Tween 80
Test Observations Specifications and Inferences
Appearance Yellowish Liquid Complied
Solubility Water soluble Complied
pH 4.6 4.5 – 5.5, Complied
Residue on Ignition 0.2% ≤0.25%
6. Carbopol 980 NF
Carbopol® 980 polymer is high molecular weight crosslinked polymer of acrylic acid,
which confirms to USP/INF specifications. Carbopol® 980 NF polymer is a highly
efficient thickener and it is ideal for formulating clear aqueous and hydroalcoholic gels.
Chapter 2: Preformulation Studies
56
Fig 2.5: Certificate of Analysis of Carbopol 980 NF
7. Carbopol 971P NF
Carbopol® 971P NF polymer is a lightly crosslinked polymer with long rheology, which
will result in flow like honey in a semisolid formulation. Carbopol 971P NF polymer can
be used to formulate low viscosity lotions, gels and liquids with good clarity.
Chapter 2: Preformulation Studies
57
Fig 2.6: Certificate of Analysis of Carbopol 971P NF
8. Carbopol 974P NF
Carbopol® 974P NF polymer is a highly crosslinked polymer and produces highly
viscous gels. Carbopol 974P NF polymer can be used to formulate viscous gels,
emulsions and suspensions.
Fig 2.7: Certificate of Analysis of Carbopol 974P NF
Chapter 2: Preformulation Studies
58
9. Transcutol P (Diethylene Glycol monoethylether) USP:
Transcutol P is a colorless liquid. It is soluble in both water and oil. It is used as a
cosurfactant, solubilizer and penetration enhancer in oral, rectal, vaginal, topical and
nasal delivery systems. It is a high performance solubilizer/solvent for many poorly
soluble compounds. It can be incorporated into all types of emulsions, solutions and gels.
10. Labrasol (Caprylocaproyl polyoxyl-8 glycerides NF)
A non-ionic water dispersible surfactant composed of well-characterised polyethylene
glycol (PEG) esters, a small glyceride fraction and free PEG. An oil-in-water surfactant
used to solubilize active pharmaceutical ingredients and promote drug penetration and
permeation.
11. Lutrol ® F68 (Poloxamer 188)
Lutrol ® F68 (Poloxamer 188) is a difunctional block copolymer surfactant terminating
in primary hydroxyl groups. It is a nonionic surfactant that is 100% active and relatively
nontoxic.
Fig 2.8: Certificate of Analysis of Transcutol P & Labrasol
Chapter 2: Preformulation Studies
59
Fig 2.9: Certificate of Analysis of Poloxamer 188 (Lutrol ® F68)
12. Oleic acid
Oleic acid is a fatty acid that occurs naturally in various animal and vegetable fats and
oils. In chemical terms, oleic acid is monounsaturated omega-9 fatty acid. Oleic acid is
used as an excipient in pharmaceuticals and as an emulsifying or solubilizing agent in
aerosol products. It is also used as penetration enhancer in topical formulations.
Table 2.12: Monographic evaluation of Oleic acid
Tests Specifications Results
Description Colorless to light yellow liquid Complied
Odor Peculiar Lard-Like odor Complied
Boiling Point 286.11°C (547°F) 285.5
Specific Gravity 0.895 0.889
13. Kollidon® 30 (Polyvinylpyrrolidone)
Polyvinylpyrrolidone (PVP), also commonly called Polyvidone or Povidone, is a water-
soluble polymer made from the monomer N-vinylpyrrolidone. When dry it is a light flaky
powder, which readily absorbs up to 40% of its weight in atmospheric water. In solution,
it has excellent wetting properties and readily forms films.
Chapter 2: Preformulation Studies
60
Fig 2.10: Certificate of Analysis of Kollidon® 30 (Polyvinylpyrrolidone)
14. Eudragit RL 100 (Ammonio Methacrylate Copolymer Type A)
Eudragit® RL is copolymer of ethyl acrylate, methyl methacrylate and a low content of a
methacrylic acid ester with quaternary ammonium groups (trimethylammonioethyl
methacrylate chloride). The ammonium groups are present as salts and make the
polymers permeable.
Chapter 2: Preformulation Studies
61
Table 2.13: Monographic evaluation of Eudragit RL 100
Tests
Specifications Results
Description Colourless, clear to cloudy granules with a faint
amine-like odour.
Complied
Solubility
Soluble in methanol, ethanol and isopropyl alcohol
Soluble in acetone, ethyl acetate and methylene
chloride to give clear to cloudy solutions.
Complied
Residue on
evaporation Not less than 97.0 % Passes
Loss on drying Max. 3.0 % 2.8
Apparent
viscosity cP Max. 15 13.9
15. Menthol
Menthol is an organic compound made synthetically or obtained from peppermint or
other mint oils. It is a waxy, crystalline substance, clear or white in color, which is solid
at room temperature and melts slightly above. Menthol has local anesthetic and counter
irritant qualities. It is also used as penetration enhancer.
Table 2.14: Monographic evaluation of Menthol
Tests Specifications Results
Description Solid crystalline, white/clear in color Complied
Odor Characteristic of menthol, camphorous Complied
Melting Point 42°C (107.6°F) 41.7
Specific Gravity 0.89 0.87
16. Eugenol
Eugenol is an allyl chain-substituted guaiacol (2-methoxyphenol). It is an active element
in clove. It is also used as penetration enhancer. Its properties make it a good local
antiseptic and analgesic. It also makes a good local anesthetic for temporary relief from
toothache pain
Table 2.15: Monographic evaluation of Eugenol
Tests Specifications Results
Description Clear or pale yellow oily liquid Complies
Odor Aromatic smell typical of cloves Complies
Solubility Slightly soluble in water and soluble in organic solvents Complies
Boiling Point 252° C 251.5
Chapter 2: Preformulation Studies
62
2.3. Container Closure System
Based on literature search on packaging systems considered for dispensing
pharmaceutical gels, we used Aluminum collapsible tubes lacquered internally and
Lamitubes (Provided as Gift sample from Essel Propack, Mumbai).
Fig 2.11: Tubes for packaging formulated Gels
2.3.1. Lamitubes
Lamitubes are more resistant to air and moisture. Lamitubes – ABL: Aluminum Barrier
Laminates is used. The multiplayer tubes (lamitube) made from laminates with
aluminium foil barrier combine the excellent barrier advantages of traditional metal tubes
& the attractive visual and tactile feel of the plastic tubes. The lamitube body and the
shoulder provide excellent barrier properties against permeation of gases like oxygen.
The tube, a soft squeezable container which can be used for gels and creams, is made of
laminated material to protect the contents. The tube is hermetically sealed and almost
sterile as a result of exposure to high temperatures during the production process. The
tube has special coatings to prevent the material from reacting with the contents. The
most important feature of these tubes is the barrier properties which keep the contents
safe from the atmosphere. With their high stability, laminated tubes are suitable for
packaging a variety of products. ABL tubes, is a customized specialty laminates, having
an aluminum foil barrier, which provides superior light, air and moisture barrier along
with reduced flavor absorption. It has tamper evident closure with nozzle seal. The
material density offers a more durable tube and allows for additional dispensing of the
products contents. Pastes, ointments, cream and gels typically dentifrice, over-the-counter
and pharmaceutical products fare well in ABL tube packaging.
These lamitubes were used for packaging and dispensing of gels and were compared with
the Aluminum collapsible tubes available as alternative packaging option.
Chapter 2: Preformulation Studies
63
2.3.2. Aluminum Collapsible Tubes
Aluminum collapsible tube is the only safe and secure means of packaging for cream,
ointment, cosmetics and other products. Aluminium tubes can be well decorated
externally and are coated internally to provide lasting stability. Impermeable nature of
aluminium collapsible tubes protects the inside product from oxidation and its collapsible
nature does not allow air contamination once opened. Aluminium collapsible tubes have
close mouth feature thus ensuring effectiveness and integrity of the product. Its seamless
and jointless nature also imparts leak proof property. Aluminum collapsible tubes of
various capacities (7 g, 10 g and 20 g) were obtained as gift sample from D. J. Industries,
Mumbai and were used for packaging and dispensing of gels.
Fig 2.12: Certificate of Compliance of Aluminum collapsible tube
Chapter 2: Preformulation Studies
64
2.4. Results
The standardization of drugs and excipients is an integral part of any research work and
ensures quality of the research outcomes.
Mepivacaine HCl and base was standardized as per the specifications given in the
monograph in USP30 NF25, 2007. Tables 2.1 and 2.2 enlist the various tests,
observations and specifications. The COA obtained from the supplier is shown in Fig 2.1.
The drug passed all the tests for identity and was well within pharmacopoeial limits. The
FT-IR spectra’s are shown in Figure 2.2
Lidocaine HCl was standardized as per the specifications given in the monograph in
USP30 NF25, 2007. Table 2.4 enlists the various tests, observations and specifications.
The drug passed all the tests for identity and was well within pharmacopoeial limits. The
FT-IR spectrum is shown in Figure 2.3.
Solvents such as Ethanol 95%, isopropyl alcohol and propylene glycol were standardized
as per the specifications of IP’2007. Tables 2.7, 2.8 and 2.9 enlist the various tests,
observations and specifications.
Polymers such as HPMC K4M, Carbopol 980 NF, Carbopol 971P NF, Carbopol 974P
NF, Kollidon® 30 and Eudragit RL 100 were standardized as per the specifications.
Table 2.10, Figures 2.5, 2.6, 2.7, 2.10 and Table 2.13 enlist the various tests,
observations and specifications respectively. The polymer passed all the tests for identity
and was well within pharmacopoeial limits.
Poloxamer F68 was standardized as per the specifications of USP’30. Figure 2.9 shows
the various tests, observations and specifications. The excipient passed all the tests for
identity and was well within pharmacopoeial limits.
Penetration enhancers such as menthol and eugenol were standardized as per the
specifications of USP’30. Tables 2.14 and 2.15 enlist the various tests, observations and
specifications. They passed all the tests for identity and were well within pharmacopoeial
limits.
Oleic acid was standardized as per the specifications of USP’30. Table 2.12 enlists the
various tests, observations and specifications. The excipient passed all the tests for
identity and was well within pharmacopoeial limits.
Chapter 2: Preformulation Studies
65
Surfactants such as Tween 80, Transcutol P and Labrasol were standardized as per the
specifications of USP’30. Tables 2.11 and Figure 2.8 enlist the various tests,
observations and specifications. The surfactant passed all the tests for identity and was
well within pharmacopoeial limits.
Packaging and dispensing tubes, lamitube and aluminum collapsible tubes of various
capacities were procured from authentic suppliers. Figure 2.12 shows the Certificate of
Compliance of Aluminum collapsible tube and its cap obtained from the manufacturer.
Lamitube of capacity 20g was used as supplied from manufacturer.
The standardized drugs and excipients were used in different permutations to develop
various formulations such as gels, nanoemulsions and metered dose topical spray
formulations as described in the following chapters.
Chapter 2: Preformulation Studies
66
2.5. Analytical Method Development and Validation
2.5.1. Introduction
Analytical method development and validation play an important role in the formulation
and manufacture of pharmaceuticals. For routine analysis of drug in respective
formulations, assessment of drug content in the developed formulations, to detect and
quantify the drug in the presence of other constituents of the formulation and
investigation of release of drug from the developed formulations, suitable analytical
method needs to be developed and validated. For this purpose following analytical
methods were developed.
2.5.2. Analytical Method Validation
Validation of analytical methodologies is widely accepted as pivotal before they are put
into routine use. A method must be tested for effectiveness and must be appropriate for
the particular analysis to be undertaken. Method validation is defined as the process of
proving, through scientific studies, that an analytical method is acceptable for its intended
use and it instills confidence that the method can generate test data of acceptable quality.
The International Conference on Harmonization has issued guidelines for analytical
method validation [ICH Q2 (R1)].
Linearity and range
Linearity defines the analytical response as a function of solute concentration and range.
It prescribes a region over which acceptable linearity, precision and accuracy are
achieved. Linearity is generally reported as the variance of the slope of the regression
line. Range is the interval between the upper and lower concentrations of solute that have
been demonstrated to be determined with precision, accuracy and linearity. The ICH
guidelines specify a minimum of five concentrations, along with certain minimum
specified ranges
Accuracy and bias
Accuracy is the measure of exactness of an analytical method, or the closeness of
agreement between the measured value and the value that is accepted either as a
conventional, true value or an accepted reference value. Bias assesses the influence of the
analyst on the performance of the method.
Chapter 2: Preformulation Studies
67
Precision
Precision quantifies the variability of an analytical result as a function of operator,
method manipulations and day-to-day environment. It is also the measure of the degree of
repeatability of an analytical method under normal operation and is expressed as the
percent RSD for a statistically significant number of samples. Precision experiments give
a good indication of the performance of the method and should be repeated regularly.
Generally, any increase of the RSD above 2.0% should be investigated. According to
ICH, three types of precision can be defined and should all be assessed as described
below.
Repeatability: It refers to the results of the method operating over a short time
interval under the same conditions (inter-assay precision). It expresses the degree of
variation arising during replicate assays performed consecutively and non-
consecutively but on the same day. Repeatability should be determined from a
minimum of nine determinations covering the specified concentration range of the
procedure.
Intermediate precision (ruggedness): It refers to the results from laboratory
variations due to random events such as differences in experimental periods, analysts
and equipment.
Reproducibility: It is an indication of the ability of the method to be transferred from
one laboratory to another.
Specificity and selectivity
A method is specific if it produces a response for only one single solute. Since it is almost
impossible to develop a chromatographic assay for a drug in a matrix that will respond to
only the compound of interest, the term selectivity is more appropriate. Selectivity
describes the ability of an analytical method to differentiate various substances in the
sample and is applicable to methods in which two or more components are separated and
quantitated in a complex matrix. It is a measure of degree of interference from such
things as other active ingredients, excipients, impurities and degradation products,
ensuring that a peak response is due only to a single component, i.e., that no co-elution
exists.
Chapter 2: Preformulation Studies
68
Limit of Detection and Limit of Quantitation
The USP requires that the limit of detection (LOD) and the limit of quantitation (LOQ)
be determined for studies that involve the detection and quantitation of components at or
near trace levels. The LOD is defined as the lowest concentration of a solute in a sample
that can be detected, though not necessarily quantitated and the LOQ is defined as the
lowest concentration of a solute in a sample that can be determined with acceptable
precision and accuracy under the stated operational conditions of the method. LOD was
based on determination of standard deviation of the response and the slope of the
corresponding curve using the following equation-
LOD = 3 s/m
Where s, the noise of estimate, is the standard deviation of the absorbance of the sample
and m is the slope of the related calibration graphs.
LOQ was based on determination of standard deviation of the response and the slope of
the corresponding curve using the following equation-
LOQ = 10 s/m
Where s, the noise of estimate, is the standard deviation of the absorbance of the sample
and m is the slope of the related calibration graphs.
2.5.3. UV–visible spectrophotometric method for estimation of Mepivacaine HCl
and Mepivacaine base for drug assay in semisolid dosage form
The technique of ultraviolet-visible spectroscopy is one of the most frequently employed
in pharmaceutical analysis. It involves the measurement of the amount of ultraviolet-
visible (190 - 600 nm) radiation absorbed by a substance in solution. Presently, the use of
ultraviolet spectrophotometry as an analytical procedure has become less popular due to
the advent of more sophisticated techniques such as chromatographic separation
techniques resulting in rare literature been available for the determination of mepivacaine
in pharmaceutical formulations. Hence, an attempt was made to develop and validate an
ultraviolet absorption spectrophotometric method for the determination of mepivacaine in
topical formulations and compare the outcomes to those obtained from HPLC.
The analytical procedure employed for quantitation of mepivacaine HCl and mepivacaine
base by U.V. spectrophotometry was determined in phosphate buffer solution (PBS) pH
6.8 and methanol.
Chapter 2: Preformulation Studies
69
2.5.3.1. Method Development:
Mepivacaine HCl and base solutions (100 μg/ml) both in methanol and phosphate buffer
solution pH 6.8 were scanned in the range of 200-400 nm using JASCO Ultraviolet
Spectrophotometer. The absorption maximum was obtained at 263 nm for phosphate
buffer pH 6.8 as well as for methanol are as shown in the figure 2.14 and 2.16. Hence
calibration curve was obtained using both the solvents. The following spectrophotometric
assay method was developed for analysis of the drug in the topical formulations:
Accurately weighed 100mg of drug was dissolved in sufficient amount of phosphate
buffer solution (PBS) pH 6.8 (or methanol) in a 100ml volumetric flask and diluted to
volume with phosphate buffer pH 6.8 (or methanol) so as to obtain solution of
concentration 1000μg/ml. From this solution, aliquots of 1.5, 2, 2.5, 3, 3.5, 4 and 4.5 ml
were withdrawn in 10 ml volumetric flasks and diluted to volume with PBS pH 6.8
solutions (or methanol) so as to obtain standard solutions of concentrations 150, 200, 250,
300, 350, 400 and 450 μg/ml respectively. The absorbance of the standard solution was
determined on U.V. Spectrophotometer at 263 nm. In order to determine the repeatability
of the method, standardization of the drug was carried out in triplicates. A standard plot
of absorbance verses concentration of drug in µg/ml was obtained. The results are as
given in Table 2.16 and 2.17.
Table 2.16: Standard calibration data for MH and MB in PBS pH 6.8 at 263 nm
Concentration (µg/ml) Absorbance at 263 nm
Mepivacaine HCl Mepivacaine Base
150 0.2297± 0.03 0.2814± 0.03
200 0.3413± 0.03 0.3889± 0.02
250 0.4174± 0.02 0.4873± 0.03
300 0.4981± 0.02 0.5915± 0.03
350 0.5585± 0.05 0.657± 0.05
400 0.6305± 0.04 0.753± 0.04
450 0.6933± 0.03 0.8419± 0.04
Chapter 2: Preformulation Studies
70
Fig 2.13: Standard Calibration curve of Mepivacaine HCl & Base in PBS pH 6.8
Fig 2.14: UV Scan of Mepivacaine HCl and Base in PBS pH 6.8
Table 2.17: Standard calibration data for MH and MB in Methanol at 263 nm
Concentration (µg/ml) Absorbance at 263 nm
Mepivacaine HCl Mepivacaine Base
150 0.2254± 0.05 0.2601± 0.05
200 0.3241± 0.05 0.3381± 0.05
250 0.3982 ± 0.04 0.3986 ± 0.04
300 0.4694 ± 0.05 0.4774 ± 0.05
350 0.5399 ± 0.03 0.5491 ± 0.03
400 0.6115 ± 0.04 0.6205 ± 0.04
450 0.6992 ± 0.03 0.7039 ± 0.03
Chapter 2: Preformulation Studies
71
Fig 2.15: Standard Calibration curve of Mepivacaine HCl and Base in Methanol
Fig 2.16: UV Scan of Mepivacaine HCl and base in Methanol
2.5.3.2. Validation of developed UV Spectrophotometric method of Mepivacaine
The spectrophotometric method selected for the analysis of mepivacaine HCl and
mepivacaine base was validated for linearity, precision, accuracy, limit of detection, limit
of quantification and specificity.
1) Linearity:
Linearity was determined by making three calibration curves. For preparation of each
calibration curve, three calibration standard solutions were prepared at concentrations
ranging from 150- 450 μg/ml of mepivacaine. Absorbance of each standard solution was
measured at 263nm. The absorbance was plotted against the corresponding
concentrations to obtain the calibration graph and was treated by linear regression
analysis. The calibration curves were found to be linear in the aforementioned
concentrations. The correlation coefficient (r²) of determination was 0.999 in phosphate
buffer. The results of test for linearity were evaluated using the regression line equation
Chapter 2: Preformulation Studies
72
obtained by the method of least squares. The ideal r² value should be more than or equal
to 0.998.
2) Accuracy:
Accuracy of the method was studied by recovery experiments. Accuracy is the percent of
analyte recovered by assay from a known added amount. Results are given in Tables 2.18
and 2.19.
Table 2.18: Intra-day and Inter-day accuracy of UV method developed for MH
Intra day Inter day
Added(μg/ml) Measured
(μg/ml) % recovery Added(μg/ml)
Measured
(μg/ml) % recovery
10 9.97 99.7 10 9.86 98.6
20 20.01 100.05 20 19.58 97.9
30 29.78 99.26 30 29.61 98.69
Table 2.19: Intraday and Inter-day accuracy of UV method developed for MB
Intra day Inter day
Added(μg/ml) Measured
(μg/ml) % recovery Added(μg/ml)
Measured
(μg/ml) % recovery
10 9.95 99.5 10 9.83 98.3
20 19.96 99.8 20 19.68 98.4
30 29.68 98.92 30 29.58 98.59
3) Precision:
It was determined by repeatability (intraday) and intermediate precision (inter-day) and
reported as % RSD for a statistically significant number of replicate measurements. The
intermediate precision was studied by comparing the assays on three different days and
the results were documented as the standard deviation and % RSD. Precision of assay
(intraday precision i.e. repeatability) was evaluated by carrying out three independent
assays of test samples of mepivacaine HCl and base. The intermediate precision (inter-
day precision) of the method was also evaluated using two different analysts, systems and
different days in the same laboratory. The results are given in Tables 2.20 and 2.21.
Table 2.20: Intraday and Inter-day precision of UV method developed for MH
Intra day Inter day
Added(μg/ml) Measured
(μg/ml) % RSD Added(μg/ml)
Measured
(μg/ml) % RSD
10 9.47 0.74 10 9.66 0.76
20 20.01 0.77 20 19.58 0.62
30 29.78 0.64 30 29.61 0.60
Chapter 2: Preformulation Studies
73
Table 2.21: Intraday and Inter-day precision of UV method developed for MB
Intra day Inter day
Added (μg/ml) Measured
(μg/ml) % RSD Added (μg/ml)
Measured
(μg/ml) % RSD
10 9.59 0.74 10 9.58 0.75
20 19.98 0.76 20 19.64 0.62
30 29.63 0.63 30 29.55 0.59
4) Limit of Detection
This is the minimum concentration of mepivacaine HCl and mepivacaine base that can be
detected but could not be quantified. The concentration at which smallest peak in the
spectrum was observed, was noted. The corresponding peak concentration of
mepivacaine HCl and mepivacaine base determined from the calibration curve was found
to be 20μg/ml.
5) Limit of Quantification
It is the minimum concentration of mepivacaine that can be quantified. The smallest peak
of mepivacaine HCl and mepivacaine base that could be detected and quantified in the
spectrum was determined. The LOQ was found to be 50μg/ml.
6) Specificity:
The specificity indicates that there was no interference observed during the test.
Table 2.22: Validation data of developed UV method for Mepivacaine HCl
Analytical Parameters Results
LOD(μg/ml) 20
LOQ(μg/ml) 60
Linearity
Range(μg/ml) 150-450
Slope ±% RSD 0.001±0.005
System precision
Amount taken(μg/ml) 10.00
Amount detected (μg/ml) ±% RSD 9.978±0.7
Chapter 2: Preformulation Studies
74
Table 2.23: Validation data of developed UV method for Mepivacaine Base
Analytical Parameters Results
LOD (μg/ml) 20
LOQ (μg/ml) 60
Linearity
Range (μg/ml) 150-450
Slope ±% RSD 0.001±0.005
System precision
Amount taken (μg/ml) 10.00
Amount detected (μg/ml) ±% RSD 9.965±0.7
2.5.4. UV–visible spectrophotometric method for estimation of Lidocaine HCl
The following spectrophotometric assay method was developed for analysis of the
Lidocaine HCl: Accurately weighed 100mg of drug was dissolved in sufficient amount of
phosphate buffer pH 6.8 in a 100ml volumetric flask and diluted to volume with
phosphate buffer pH 6.8 so as to obtain solution of concentration 1000μg/ml. From this
solution, aliquots of 1.5, 2, 2.5, 3, 3.5, 4 and 4.5 ml were withdrawn in 10 ml volumetric
flasks and diluted to volume with phosphate buffer pH 6.8 solutions so as to obtain
standard solutions of concentrations 150, 200, 250, 300, 350, 400 and 450μg/ml
respectively. The absorbance of the standard solution was determined on U.V.
Spectrophotometer at 264 nm. In order to determine the repeatability of the method,
standardization of the drug was carried out in triplicates. A standard plot of absorbance
verses concentration of drug in µg/ml was obtained. The results are as tabulated.
Fig 2.17: UV Scan of Lidocaine
HCl in PBS pH 6.8 Fig 2.18: Standard Calibration
curve of Lidocaine HCl
Chapter 2: Preformulation Studies
75
Table 2.24: Standard calibration curve for Lidocaine in PBS pH 6.8 at 264 nm
Concentration (µg/ml) Absorbance at 263 nm
150 0.2677
200 0.3589
250 0.4468
300 0.5362
350 0.6111
400 0.7050
450 0.7832
2.5.5. High Performance Liquid Chromatography (HPLC) Method for Estimation
of Mepivacaine HCl and Mepivacaine Base
HPLC provides rapid high resolution of compounds resulting in an efficient method of
analysis which can be completed over relatively short periods of time. In addition to this,
coupling HPLC to suitable detection methods provides increased sensitivity and
selectivity to facilitate accurate, precise and reproducible analysis of pharmaceutical
products. HPLC technique is based on the separation of components of a mixture by
virtue of differences in the equilibrium distribution of the components between
stationary and the mobile phase. The compound partitions between the different phases
based on their physicochemical properties and affinity for either of the phases. This
results in compounds eluting at different times and the consequent separation of the
drugs and components. The design of a successful HPLC separation method depends on
matching the right mobile phase to a given column and sample. Solvents used should be
readily available, compatible with the detector, safe to use, pure and relatively
unreactive. The solvent should be able to dissolve the sample.
For determination of concentration of Mepivacaine (Base & HCl) in formulation and in
skin layers, HPLC was performed using mobile phases as reported in the literature [Gill
R; 1984, GuCvello P. L; 1993, Einosuke T; 2006, Arie A.V; 1996].
Chapter 2: Preformulation Studies
76
Table 2.25: Mobile phases tried for HPLC analysis of Mepivacaine
Sr
No
Wavelength of
detection
Flow rate
(ml/min) Mobile Phase
1 210 nm 1.1
6.8:93.2 (v/v) isopropanol-sodium hydrogen
phosphate buffer solution with the pH adjusted to
6.8 using phosphoric acid.
2 210 nm 1.0 acetonitrile–methanol–30mM NaH2PO4 (pH 5.6)
(1:1:3, v/v/v).
3 210 nm 1.0 acetonitrile–methanol–30mM NaH2PO4 (pH 5.6)
(1:1:3: 0.05, v/v/v).
4 205 nm 1.0 0.01 M sodium dihydrogenphosphate (pH 2.1)-
acetonitrile (80:20, v/v).
5 210 nm 1.0 Methanol: water: 1% v/v solution of phosphoric
acid: hexylamine (30:70:100:1.4, v/v)
Acetonitrile and methanol, the two commonly used solvents, have ultraviolet cut-off
wavelength values of 190 and 205 nm respectively when highly purified. However the
peak obtained for mepivacaine was not distinct and poor resolution was obtained. Hence
mobile phase was developed for mepivacaine HPLC analysis. Sharp peaks that were well
resolved from the solvent front were observed with mobile phases comprising acetonitrile
and phosphate buffer solution pH 6.8 value.
2.5.5.1. Analytical Method Development for Assay of Mepivacaine
The aim of the investigation was to develop a method for the assay of drugs, Mepivacaine
HCl and Mepivacine base from the developed topical formulations.
Chromatographic conditions
An overview chromatographic condition of the HPLC method for Mepivacaine
quantification is as follows:
Chapter 2: Preformulation Studies
77
Column C18 Gold column (46mm x 250 mm) 2.5 µm
Mobile Phase Acetonitrile: Phosphate Buffer Solution pH 6.8 (6:4)
Flow rate 1 ml/min
Run time 15 min
Injection volume 20 µl
Mepivacaine quantification
wavelength 210 nm
Calibration curve for Mepivacaine HCl & Base: - Mepivacaine HCl or base (10mg)
was taken in 100 ml volumetric flask and diluted to volume with phosphate buffer
solution pH 6.8 and used as stock solution for preparation of calibration curves. The
reference solution was diluted with PBS to obtain concentration ranges of Mepivacaine
HCl/base (0.5-5 µg/ml). The calibration curves were plotted as peak area vs.
concentration (Fig. 2.6.9 & 2.6.10).
Table 2.26. Standard calibration data of MB in PBS pH 6.8
Concentration
(µg/ml)
Area
Mepivacaine HCl Mepivacaine Base
0 0 0
0.5 30.57 31.26
1 65.32 68.54
2 121.39 125.78
3 187.34 191.32
4 241.28 250.11
5 299.12 302.76
Fig 2.19: HPLC Scan of MH in
PBS in pH 6.8
Fig 2.20: HPLC Scan of MB in PBS in
pH 6.8
Chapter 2: Preformulation Studies
78
2.5.5.2. Analytical Method Development and Validation for Quantification of
Mepivacaine from In vitro diffusion media
Selection of suitable drug diffusion media and development of an analytical method for
quantification of mepivacaine from the diffusion media: Suitable diffusion media is the
pre requisite for the in vitro diffusion studies. Since the formulations are intended for
topical applications, the receptor media was selected such that its pH simulates to the skin
pH. Phosphate buffer solution of pH 6.8 was used as the receptor media for in vitro
diffusion studies.
The objective of validation was to demonstrate the suitability of developed method for
quantification of drug from the in vitro diffusion media i.e., the method should be
sensitive enough to detect low concentrations and should be reproducible and linear. The
method was validated for the following parameters.
A. Linearity
The linearity of the method was investigated by studying the linear regression of the areas
under the peaks obtained by analyzing solutions with mepivacaine concentrations in the
range of 1.0 – 10.0 µg/mL.
B. Precision
Intra-day precision: The intraday precision was evaluated (n = 3) at three
concentration levels (1.0, 5.0 and 10.0 µg/mL) of mepivacaine and % relative standard
deviation of three values was calculated.
Fig 2.21: Standard Calibration curve of
MH in PBS pH 6.8
Fig 2.22: Standard Calibration curve of
MB in PBS pH 6.8
Chapter 2: Preformulation Studies
79
Inter-day precision: Concentrations of 1.0, 5.0 and 10.0 µg/mL of mepivacaine were
analyzed on three different days for assessing inter day precision. Three triplicates per
concentration were injected into the HPLC.
Table 2.27:Statistical data showing intra-day precision of method for quantitation of
mepivacaine during in vitro diffusion study.
Concentration
µg/mL
Average
area
Predicted
average
% RSD SE R2
Day 1
0.1 102.59 102.65 0.1456 0.1412 0.9998
0.6 321.89 321.90 0.0765 0.2341
1.0 609.98 610.21 0.0165 0.2781
Day 2
0.1 103.27 103.30 0.2134 0.2121 0.9999
0.6 322.45 322.50 0.0856 0.3843
1.0 610.32 610.45 0.0201 0.3118
Day 3
0.1 102.98 103.45 0.2351 0.3138 0.9999
0.6 322.88 323.05 0.0961 0.3112
1.0 610.76 610.95 0.0271 0.2890
Table 2.28: Statistical data showing inter-day precision of method for quantitation of
mepivacaine during in vitro diffusion study
Concentration
µg/mL
Average
area
Predicted
average % RSD R
2
0.1 102.59 103.05 0.1027
0.9999 0.6 321.89 322.15 0.0573
1.0 609.98 610.12 0.0654
2.5.5.3 Method Development and Validation for Quantification of Mepivacaine from
Skin Homogenate.
Measurement of drug concentration in the skin is essential during the development of
topical products. Active accumulation in the skin with minimal or no systemic absorption
is desired. This requires the development of a robust and sensitive method to extract and
quantify the drug in the skin in order to predict skin targeting. An analytical method was
developed and validated for quantification of mepivacaine in porcine and rat skin samples
for ex vivo and in vivo penetration studies. Porcine ear skin was used for ex vivo skin
penetration studies. Therefore initial method development was carried out with porcine
Chapter 2: Preformulation Studies
80
ear skin. Further the method was extended to rat skin homogenate and standard curves
were prepared in homogenates of rat skin layers (epidermis and dermis).
The aim of this part of research work was to develop a simple method to extract and
quantify mepivacaine from porcine and rat skin samples in order to predict if adequate
amounts of active have reached viable layers of skin and to assay the drug using HPLC
method of analysis.
Selection of Internal Standard: Internal standards are commonly used in quantitative
bioanalysis particularly in HPLC / MS based bio-analysis. By finding a good internal
standard and using analyte / IS response ratios for quantitation, variations in absolute
responses other than those related to analyte concentration could be corrected, which help
maintain the accuracy of quantitative results. Various chemical compounds were
investigated for their suitability as internal standard along with mepivacaine.
Method development for extraction of mepivacaine from skin homogenate: The
method was developed in model of porcine ear skin, as porcine ear skin is well accepted
as a decent facsimile of the human counterpart because of its physiological and structural
similarity to human skin [Schmook FP; 2001; Herkenne C; 2008, Teichmann A ; 2006].
A) Skin preparations: Porcine ear skin
The porcine ears were obtained post sacrifice from the local abattoir (VileParle,
Mumbai), before the pig carcass was exposed to the normal high temperature cleaning
procedure. The ears were stored at -20oC and thawed slowly at 4
oC before gently cutting
the hair and removing full thickness skin from the back of the ears using a scalpel without
damaging or scratching the surface in order to ensure the integrity of the skin barrier.
Subcutaneous tissue was carefully removed with a scalpel, and the skin was cut into
appropriate pieces and was stored at -20oC until further use (Not more than 2 weeks).
B) Extraction of drug from skin homogenate
Mepivacaine was extracted from the skin by cutting it into very fine pieces using scissors
and homogenized with tissue homogenizer separately in methanol (5ml) as mepivacaine
has very good solubility in methanol. The homogenate was then vortex mixed and further
homogenized by ultra sonication for complete extraction of actives. The resulting
homogenate was filtered using 0.2µ membrane filter to get clear solution and active
content in the filtrate was determined after appropriate dilution.
Chapter 2: Preformulation Studies
81
In order to confirm the reliability, accuracy and precision of the method, several
parameters were investigated according to ICH guidelines for analyzing mepivacaine in
porcine ear skin samples.
A. Specificity
The specificity of the method was verified by evaluating the interference of skin
components with the assay of active, mepivacaine. To determine the method specificity,
the control skin samples were treated by the same procedure as that to extract actives and
the spectra obtained from these injections were compared to the skin homogenate spiked
with mepivacaine.
B. Linearity
Linearity curves to estimate mepivacaine in skin samples were constructed by assaying
skin homogenates spiked with mepivacaine in the concentration range of 2.0 to 20 µg/ml.
These standard solutions were vortexed for 30 secs and centrifuged at 3000 rpm for
10min. The supernatants were collected and then evaporated. The residue collected after
evaporation was reconstituted to 10 mL with mobile phase. The diluted samples were
filtered; injected into the HPLC and chromatograms were recorded at 210 nm. An
appropriate concentration of internal standard was selected for spiking the homogenates
based on preliminary studies. The final solutions were injected in triplicate. The
calibration curve was obtained by plotting peak area ratio of mepicavaine v/s
concentration. The linearity was repeated on three different days. The regression
coefficient was calculated for each day.
C. Precision
Intra-day precision
The intra-day precision was determined by sequential analysis at three concentration
levels of mepivacaine (2.0, 10.0 & 20 µg/mL). Each sample was analyzed at three
different times a day, totaling to nine analyses in a day.
Inter-day precision
Three different concentrations 2.0, 10.0 & 20 µg/mL of mepivacaine were analyzed on
three different days. Three replicates per concentration were injected in to the HPLC. The
SD and standard error of mean (SE) were compared as a measure of scatter of method
precision.
Chapter 2: Preformulation Studies
82
Repeatability
System repeatability was determined by injecting 10 replicates of 4.0 µg/mL and 6.0
µg/mL of mepivacaine solution respectively which were prepared by spiking the
appropriate concentrations of drug and internal standard into the skin homogenate
followed by extraction. The entire procedure was repeated on three different days and
the relative standard deviation (% RSD) was calculated to determine the deviation of
each measurement from mean value for each day.
Recovery
High recovery of the drug from the homogenate matrix is a desirable outcome of sample
preparation and is therefore an important characteristic of the extraction procedure. The
absolute recovery was determined as the ratio of response measured for the spiked
sample (in homogenate matrix) treated according to the extraction procedure to that of a
non biological sample spiked with the same quantity of the drug and directly injected into
the chromatographic system. Three different concentration levels (2.0, 10.0 and 20.0
µg/mL) were investigated for determination of extraction recovery.
Fig 2.25: Calibration Curve of MB in the presence of porcine skin matrix
Table 2.29: Statistical data showing intra-day precision for extraction of mepivacaine
from porcine ear skin
Fig 2.23: Chromatogram showing analysis
of blank porcine ear skin homogenate.
Fig 2.24: Chromatogram showing
analysis of drug loaded porcine ear
skin homogenate.
Chapter 2: Preformulation Studies
83
Concentration
µg/mL
Average
area
Predicted
average
% RSD R2
Day 1
2.0 0.3612 0.3620 0.3882 0.9998
8.0 1.3789 1.3795 0.2291
10.0 2.5871 2.5882 0.2339
Day 2
2.0 0.3725 0.3730 0.3551 0.9996
8.0 1.3890 1.3895 0.2392
10.0 2.5915 2.5928 0.2531
Day 3
2.0 0.3911 0.3925 0.3882 0.9997
8.0 1.3995 1.4004 0.2748
10.0 2.5976 2.5985 0.2411
Table 2.30: Statistical data showing inter-day precision for extraction of mepivacaine
from porcine ear skin
Concentration
µg/mL
Average
area
Predicted
average
SD % RSD R2
2.0 0.3512 0.3555 0.0035 0.9862 0.9987
8.0 1.3201 1.3236 0.0193 1.0931
10.0 2.5780 2.5809 0.0249 0.8947
Table 2.31: Statistical data showing repeatability of mepivacaine from porcine ear skin
Average Area Predicted Area SD % RSD
Day 1 0.6592 0.6389 0.0045 0.7375
Day 2 0.6639 0.6385 0.0049 0.7278
Day 3 0.6712 0.6321 0.0048 0.6911
Table 2.32: Recovery data of mepivacaine in spiked porcine skin samples
Conc
µg/mL
Non biological Spiked
sample (µg/mL)
Tissue homogenate
matrix
%
Recovery
2.0 0.4331 0.4550 96.34
8.0 1.3254 1.3431 98.45
10.0 2.6125 2.6250 97.59
Chapter 2: Preformulation Studies
84
Table 2.33: Validation parameters of the developed HPLC method using porcine skin
Analytical Parameters Results
LOD (ng/ml) 20
LOQ (ng/ml) 60
Linearity
Range (ng/ml) 50-500
Regression values 0.9992
Demonstration of suitability of extraction method for quantification of mepivacaine
from rat skin:
A. Skin preparation: Rat skin was depilated to remove hair. The skin was excised and
carefully dissected making sure that the subcutaneous fat was maximally removed and
stored at -40oC until further use.
B. Extraction of drug from skin homogenate: The active mepivacaine from the rat skin
layer (epidermis and dermis) homogenate was extracted using the same method as
described in the section of porcine ear skin.
C. Validation of the developed method
Validation of method for extraction of mepivacaine from rat skin layers (epidermis and
dermis) was carried out as described under previous section. The amount of active in
stratum corneum was analyzed by tape stripping.
Fig 2.28: Calibration Curve of MB in the presence of rat skin matrix.
Fig 2.26: Chromatogram showing
analysis of blank rat skin homogenate
Fig 2.27: Chromatogram showing analysis
of drug in rat skin homogenate
Chapter 2: Preformulation Studies
85
Table 2.34: Validation parameters of the developed HPLC method using rat skin matrix
Analytical Parameters Results
LOD (ng/ml) 20
LOQ (μg/ml) 60
Linearity
Range (ng/ml) 50-500
Regression values 0.9992
2.5.5.4 Method Development and Validation for drug extracted from Tape Strips
used for in vivo Penetration Studies
Sequential tape-stripping of the stratum corneum (SC) of skin allowed horizontal
fractions of membrane to be obtained. The method involves extracting the tape strips in
suitable solvent so as to recover and quantify the absorbed active. The method of
quantification depends on the nature of the active and the amount present on the tape
strips. The key criteria’s are that the extraction process should be efficient and
reproducible, and that it should be free from interference from components of the SC
and/or the tape adhesive. Therefore the aim was to develop and validate a standardized
method for assessment of active compound from the skin by tape stripping method and
HPLC analysis procedure which should be sensitive enough for determination of active in
extraction samples from adhesive tapes.
Development of extraction procedure of mepivacaine from tape strips:
Selection of adhesive Tapes: Selection of adhesive tape with appropriate properties is a
necessary prerequisite for accurate studies based on stratum stripping for determination
of an active in the stratum corneum. The prerequisite of the tape used in the present
experimental part was to show no absorbance at wavelength used for quantification of
mepivacaine thus preventing interferences.
Extraction Procedure: To determine HPLC specificity, the tapes were extracted with
5ml extraction medium in a stoppered 25 ml conical flask. The contents were then
sonicated and filtered. The filtered aliquot solution was injected in to the HPLC column
without further dilution. The chromatograms obtained from these injections were
compared with those obtained for the standard drug solution.
Chapter 2: Preformulation Studies
86
Development of extraction procedure from 3M Micropore tape strips:
Sr No Extraction procedure from 3M Micropore tape strips
1. Tape strips were immersed in chloroform, sonicated and the supernatant
was filtered through 0.2µ membrane filter and injected into HPLC
2. Tape strips were immersed in methanol, sonicated and the supernatant was
filtered through 0.2µ membrane filter and injected into HPLC
3. Tape strips were immersed in IPA, sonicated and the supernatant was
filtered through 0.2µ membrane filter and injected into HPLC
Sample Preparation: The extraction procedure was validated by spiking tape stripped
samples of untreated SC with known amounts of active chosen to represent the expected
range of concentrations encountered during the in vivo experiments.
A. Specificity
To assure that there is no interference of matrix constituents of the tape (e.g. adhesive,
polymeric film on the tape) and skin components (such as lipids, proteins) with the active
drug peak, tapes stripped from untreated rat skin were compared with the tape stripped
from the untreated skin spiked with standard mepivacaine solution.
B. Precision
Inter day variation: The precision under the same operating conditions over a short
interval of time was determined by sequential analysis of spiked solutions at three
concentration levels (0.5, 1.5 and 2.5 µg/ml). Each sample was analyzed at three different
times of a day, totaling to nine analyses a day.
Inter day variation: Three replicates of three concentration levels 0.5, 1.5 and 2.5 µg/ml
were analyzed on three separate days for the estimation of intermediate precision and the
measure of scatter of method was computed.
System repeatability: Tapes were spiked with mepivacaine solution to yield loading dose
of 1.0 µg/ml on the tapes. The samples were injected six times and the chromatograms
were recorded. The experiment was repeated on three different days and the relative
standard deviation was calculated for each day. The repeatability data is shown in Table
2.37.
Chapter 2: Preformulation Studies
87
C. Linearity
Linearity of the analytical method was determined by spiking the tape strips with stock
solutions of mepivacaine doses in the range of 0.5 to 5.0 µg/mL.
D. Recovery
The demonstration of the method’s extraction efficiency was achieved using the response
measured for the spiked sample (skin stripped tapes) treated according to the whole
analytical procedure to that of a non biological sample spiked with the same quantity of
the drug and directly injected into the chromatographic system. Three different
concentration levels (0.5, 1.5 and 2.5 µg/mL) were investigated for determination of
extraction recovery. Results are as in Table 2.38
Fig 2.31: Calibration Curve of MB in the presence of tape strip sample
Fig 2.29: Chromatogram showing peaks
of blank tape strip sample
Fig 2.30: Chromatogram showing
peaks of MB+IS in presence of tape
strip sample
Chapter 2: Preformulation Studies
88
Table 2.35: Statistical data showing intra-day precision of method for extraction of
mepivacaine from tape strip
Concentration
µg/mL
Average area Predicted
average
% RSD R2
Day 1
0.5 625.21 625.56 0.4362 0.9998
1.5 1477.12 1478.67 0.3982
2.0 2832.67 2840.50 0.3313
Day 2
0.5 627.45 627.45 0.4578 0.9997
1.5 1480.49 1480.49 0.3321
2.0 2838.46 2838.46 0.2291
Day 3
0.5 631.87 632.45 0.4118 0.9999
1.5 1488.10 1489.22 0.3391
2.0 2845.39 2848.21 0.2274
Table 2.36: Statistical data showing inter-day precision of method for extraction of
mepivacaine from tape strips
Concentration
µg/mL Average area Predicted average % RSD R
2
0.5 620.21 621.50 0.5690
0.9997 1.5 1480.39 1485.12 0.6112
2.0 2898.54 2899.79 0.5331
Table 2.37: Statistical data showing repeatability of method for extraction of mepivacaine
from tape strips
Conc µg/mL Average Area Predicted Area % RSD
Day 1 1.0 1175.36 1175.89 0.7762
Day 2 1.0 1178.43 1178.50 0.8112
Day 3 1.0 1180.12 1180.26 0.7743
Table 2.38: Recovery data for estimation of mepivacaine in spiked tape strip samples
Conc
µg/mL
Non biological Spiked
sample (µg/mL)
Tissue homogenate
matrix
%
Recovery
Average %
recovery
0.5 621.45 621.65 98.34
97.42 1.5 1483.48 1483.60 97.47
2.0 2894.34 2895.23 96.45
Chapter 2: Preformulation Studies
89
2.5.5.5 Method Development and Validation of Mepivacaine extracted from Plasma
Measurement of drug concentration in skin is essential in the development of topical
formulations. Active accumulation in the skin with minimal or no systemic absorption is
desired. The aim of this part of the research work was to develop a sensitive and reliable
bioanalytical HPLC method for the estimation of mepivacaine in rat plasma during
pharmacokinetic studies. Thus the purpose of this work was to develop and validate the
method for extraction of the actives from plasma to prove that the pharmacological effect
produced was due to local action of the actives and no significant systemic absorption
was observed.
Preparation of Standard Solutions: The stock (100 µg/mL) and sub-stock solutions
containing mepivacaine with concentrations of 1, 5, 10, 15 and 20 µg/mL each were
prepared using methanol as solvent. Internal standard stock solution was also prepared at
a concentration of 5 µg/mL using the same solvent.
Development of Extraction Procedure for Extraction of mepivacaine from Plasma:
The purpose was to develop suitable extraction procedure for the active and the Internal
standard (IS) from plasma. Mepivacaine was separated from the plasma matrix using
liquid–liquid extraction with an organic solvent under alkaline conditions. Plasma (0.5
ml) spiked with pre determined quantity of drug solution (or IS) was mixed with
sufficient quantity of alkaline (NaOH) solution, and organic solvent (DCM). The samples
were vortexed for 20 secs, followed by centrifugation at 2000 rpm for 3 minutes. The
upper aqueous layer was aspirated to waste. The organic layer was transferred into a
conical centrifuge tube and evaporated to remove the traces of organic phase till
completely dry. The samples were reconstituted in acetonitrile to dissolve the residue.
The final concentration of drug was 100, 150, 200, 250, 300, 350, 500 ng/mL and 100
µg/mL of IS in these solutions. The solution was filtered, injected into HPLC and the
chromatograms were recorded at 210 nm.
A. Linearity
It was determined by spiking the plasma sample with stock solution of mepivacaine in the
range of 0.05 to 0.5 µg/ml and with appropriate concentration of Internal standard. The
drug was extracted and analyzed as described earlier. The calibration curve was obtained
from the least square linear regression. The regression line was used to calculate the
Chapter 2: Preformulation Studies
90
respective concentrations of drug in the plasma obtained from the rats 24 h after
application of topical dosage forms on the rats skin surface.
B. Recovery
The demonstration of the method’s extraction efficiency was achieved using the response
measured for the biological sample spiked treated according to the whole analytical
procedure to that of a non biological sample spiked with the same quantity of the active
and injected into the chromatographic system. For determination of extraction recovery
three different concentration levels, (0.08, 0.12 and 0.22 µg/ml) were studied.
C. System precision
It was determined by performing six repeated analysis of working standard solution of a
concentration of 100ng/mL. Intermediate precision was assessed by analyzing three
replicates of reference solutions at three levels (0.08, 0.12 and 0.22 µg/mL) on the same
day and on three consecutive days.
D. Accuracy
The accuracy of the analytical method was determined by replicate analysis of three
quality control samples containing known amount of analyte (0.08, 0.12 and 0.22
µg/mL).
Fig 2.34: Calibration Curve of MB in the presence of rat plasma as biological matrix.
Fig 2.33: Chromatogram showing analysis
of drug in presence of plasma.
Fig 2.32: Chromatogram showing peaks
of blank plasma sample
Chapter 2: Preformulation Studies
91
Table 2.39: Validation parameters of the developed HPLC method using rat plasma as
biological matrix
Analytical Parameters Results
LOD (ng/ml) 20
LOQ (ng/ml) 60
Linearity
Range(ng/ml) 50-500
Regression values 0.9992
System precision
Theoretical peak area ratio 2.93
Observed peak area ratio 2.89
% RSD 0.98
Table 2.40: Precision and accuracy results for developed HPLC method for mepivacaine
using rat plasma as biological matrix
Sample
n=3
Theoretical
conc
(ng/mL)
Intra-day
precision
(% RSD)
Inter-day
precision
(% RSD)
Percent
Accuracy
Mean %
Accuracy
+ % RSD
%
Recovery
Mean %
Recovery
+ %RSD
Low
Q.C 80 0.63 1.51 99.02
99.67 ±
0.70
99.45
99.45 ±
0.60
Medium
Q.C 120 0.27 1.38 100.63 98.86
High
Q.C 220 0.47 0.45 99.36 100.03
*Q.C - Quality Control
Chapter 2: Preformulation Studies
92
2.5.6. Results and Discussion:
2.5.6.1. UV–visible spectrophotometric method for estimation of Mepivacaine HCl,
Mepivacaine base and Lidocaine HCl for drug assay in semisolid dosage forms
In the present work, U.V spectrophotometric method for the quantitation of Mepivacaine
HCl and base and Lidocaine in topical dosage form was developed for routine analysis.
The method was developed by using phosphate buffer solution pH 6.8 and methanol as
solvents as the drug showed good solubility in both the solvent systems. In proposed
method, absorption maxima was obtained at 263 nm for mepivacaine base & HCl and
264 nm for lidocaine and the calibration curve obeyed Beer-Lambertz law in the
concentration range of 100-450 μg/ml with correlation coefficient (r²) of 0.9999 and
0.9998 in phosphate buffer pH 6.8 and methanol respectively. The developed method was
validated according to ICH guidelines for validation of analytical procedures. Limit of
detection was found to be 20 μg/ml and limit of quantification was 60 μg/ml for
mepivacaine HCl and base. The low values of percentage relative standard deviation
showed that the developed method was precise. All statistical data proved validity of
proposed method, which can be applied for assay of mepivacaine. Although the proposed
method was found to be linear, precise and accurate, it is not very sensitive for the
quantification of mepivacaine from in vitro/ex vivo diffusion media. Hence a more
sensitive HPLC method was developed for the analysis of mepivacaine HCl and base
from topical formulations.
2.5.6.2. Analytical Method Development and Validation for Quantification of
Mepivacaine from In vitro diffusion media
The initial mobile phases tried were based on published data on mepivacaine as shown in
Table 2.25. However a well resolved peak of drug could not be obtained. The developed
mobile phase used for the quantitation of mepivacaine from the diffusion media consisted
on acetonitrile: phosphate buffer solution pH 6.8 (6:4 v/v). The validation was carried out
to demonstrate the suitability of the developed method for quantitation of mepivacaine
from the in vitro diffusion media i.e., the method should be sensitive enough to detect
low concentrations of the active and should be repeatable and linear. The analytical
method was validated for linearity and precision.
Chapter 2: Preformulation Studies
93
Linearity: The linear regression coefficients for the constructed calibration curves of
mepivacaine demonstrated linearity with r2
value greater than 0.999.
Precision: Intra-day and Inter-day precision of mepivacaine analyzed at three different
concentrations showed % RSD values < 2 as shown in Table 2.27 and 2.28. This
indicated that the developed method for quantification of drug from the in vitro diffusion
media was precise.
Thus an analytical method for in vitro diffusion studies was developed and found to be
selective, sensitive, linear and precise.
2.5.6.3. Analytical Method for extraction of drug from Porcine Ear Skin.
Mepivacaine could be easily extracted from the porcine ear skin using tissue
homogenizer for homogenizing the skin sample and methanol as solvent for extraction
since the drug has excellent solubility in methanol. The suitability of the method was
further verified by performing the detailed validation of the method as per ICH
guidelines.
Specificity: The control skin sample HPLC spectra were compared with HPLC spectra
from mepivacaine spiked skin sample. The retention time of the drug was recorded at 5.8
min. In the chromatogram of the skin extracts, skin components did not interfere with the
peak of interest (Fig 2.23 and 2.24). Since no interference between the drug and skin
matrix components was observed in the HPLC spectra, the method was proved to be
selective and specific.
Linearity: Calibration curve constructed for mepivacaine by plotting the graph of
concentration versus mepivacaine area was found to be linear in the range of 2.0 to
10µg/ml as shown in the Fig 2.25. The analytical method showed a regression coefficient
greater than 0.999 on all the three days. Thus the linear regression analysis demonstrated
acceptability of the method for quantitative analysis of mepivacaine in the skin samples.
Intra-day and Inter-day Precision: The observed lower values of relative standard
deviation, lower % RSD values <2, at both, intra-day and inter-day analysis indicated the
Chapter 2: Preformulation Studies
94
method to be precise. It showed the acceptability of the method with adequate intra-day
and inter-day precision (Table 2.29 & 2.30).
Repeatability: SD, % RSD and SE displayed low variance for three separate days for
mepivacaine as shown in the Table 2.31. This demonstrates the method to be repeatable
for the analysis of mepivacaine from the skin homogenate.
Recovery: The recovery was calculated from the mepivacaine concentration with the non
skin sample and compared with the spiked skin homogenate. The mean recovery of
mepivacaine was 96.34%, 98.45% and 97.59% at concentrations of 2.0, 8.0 and 10.0
µg/mL respectively (Table 2.32). The average recovery over the entire analytical range
was 97.46%. From the recovery rates, it can be concluded that the extraction procedure
provided a reliable quantitative determination of the drug in skin extracts.
2.5.6.4. Analytical Method for extraction of drug from Rat Skin.
The linear regression coefficients and calibration curves in the range of 0.1 to 0.5 µg/mL
of mepivacaine showed that the extraction method from epidermis and dermis of rat skin
homogenate is linear with R2
value greater than 0.998 on three separate days. The
chromatogram showing analysis of blank and drug in rat skin homogenate are shown in
Figs 2.26 and 2.27 and the standard calibration curve of MB in the presence of rat skin
matrix is shown in Fig 2.28.
2.5.6.5. Analytical Method for determination of drug content from Tape Strips.
The 3M Micropore tapes were investigated with the aim to extract the drug efficiently
and selectively and to eliminate the interference of tape constituents such as adhesives
and polymer with the peak of interest. When Chloroform and isopropyl alcohol was used
as extraction media, interference with the active peak was observed. However, when
methanol was used as extraction media, no interference due to tape strip constituents and
skin components was observed at the Rt of the active, mepivacaine.
Specificity: The method developed here proved to be selective since the retention times
for other compounds present in the skin or in the adhesive tape, analyzed under the same
chromatographic conditions, was not similar to those obtained for mepivacaine showing
Chapter 2: Preformulation Studies
95
no interference between them and the tape matrix constituents or the skin components,
thereby validating the specificity of the technique as shown in the Figs 2.29 and 2.30.
Intra-day and Inter-day Precision: The observed lower values of relative standard
deviation, lower % RSD values <2, at both, intra-day and inter-day analysis indicated the
method to be precise. It showed the acceptability of the method with adequate intra-day
and inter-day precision (Tables 2.35 & 2.36).
Repeatability: Table 2.37 shows lower % RSD values for mepivacaine thus confirming
that the method could be repeatable and reproducible and the precision was found to be
acceptable for analytical purposes.
Linearity: Chromatograms recorded over a concentration range of 0.5 to 5.0 µg/mL
yielded a linear relationship between the peak area and the concentrations. The method
demonstrated the acceptable linearity with regression coefficient greater than 0.996. The
typical calibration curves obtained for mepivacaine are shown in Fig 2.31.
Recovery: The mean recovery for mepivacaine from the tape strips was found to be
98.34% at a concentration of 0.5 µg/mL, 97.47% at a concentration of 1.5 µg/mL and
96.45% at a concentration of 2.0 µg/mL.
Therefore, the average % recovery over the entire analytical range was 97.42. The
recovery data is summarized in Table 2.38. High recovery of the drug from the tissue
matrix is a desirable outcome of sample preparation and is therefore important
characteristic of extraction procedure. From the recovery rates, it could be concluded that
the extraction procedure provides a reliable quantitative determination of drug in tape
extracts.
2.5.6.6. Analytical Method for extraction of drug from Plasma.
Mepivacaine was extracted from the plasma matrix using liquid–liquid extraction using
dichloromethane as an organic solvent under alkaline conditions.
Linearity: Chromatograms recorded over a concentration range of 0.05 to 0.5 µg/mL
yielded a linear relationship between the peak area and concentration. The method
Chapter 2: Preformulation Studies
96
demonstrated acceptable linearity with regression coefficient greater than 0.998 as shown
in the Fig 2.34.
Recovery: The recovery study of mepivacaine from the plasma samples was studied at
three concentrations of 0.08 µg/mL, 0.12 µg/mL and 0.22 µg/mL and the mean recovery
was found to be 99.45%, 98.86% and 100.03% respectively. From the recovery data of
mepivacaine as depicted in Table 2.40, it could be concluded that this extraction
procedure was efficient and gave good recoveries.
The LOD and LOQ of mepivacaine in rat plasma matrix were found to be 20 and 60
ng/mL with mean % accuracy of 99.67%. Hence, the analytical method developed and
validated for the quantitation of mepivacaine in rat plasma matrix was sensitive, precise
and accurate.
This developed and validated HPLC method will be useful for estimation of drug from
topical formulations.
Chapter 2: Preformulation Studies
97
2.6. Forced Degradation Studies on Mepivacaine
The potential impurities of Mepivacaine HCl reported are 2, 6-Picoloxylidide
(intermediate) and -2,6-Pipecoloxylidide (side product). The Degradation impurities of
mepivacaine reported are Xylidine (Hydrolysis product), -1-Methyl-2-
piperidinecarboxylic acid (Hydrolysis product).
There is an increased interest in the identification and control of potentially genotoxic
impurities. In 2007, the European Medicines Agency (EMEA) issued guidelines on the
limits of genotoxic impurities [EU EMEA/CHMP Guidelines], and the Food and Drug
Administration (FDA) issued draft guidance on the same subject in December 2008 [US
FDA/CDER, Guidance]. A validated analytical procedure was used which was capable of
determining mepivacaine in the presence of formulation excipients and potential
degradation products was used.
2.6.1. Standard preparation
About 10 mg of mepivacaine reference standard was accurately weighed and dissolved in
the mobile phase to make 100 ml solution. A 10-ml aliquot of the resulting solution was
further diluted to 100 ml with mobile phase.
2.6.2. Test preparation
About 500-mg, accurately weighed, portion of the mepivacaine gel was dispersed by
sonication for 10 min in 50 ml of mobile phase and diluted to 100 ml with the mobile
phase. The solution was filtered using a 0.45-µm filter membrane discarding the first 15
ml. Samples of the mepivacaine gel and placebo gel were stressed under the following
conditions, and test preparations were prepared using the stressed samples.
2, 6-Picoloxylidide 2, 6-Pipecoloxylidide
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2.6.3. Thermal degradation
Mepivacaine gel was weighed and placed in a sealed glass tube. The glass tube was
placed into a heated tube furnace (Lindberg/Blue) at 150◦C for 4 hrs. The glass tube was
then allowed to cool to room temperature. Then the sample was analyzed by HPLC.
2.6.4. Photo-degradation
Mepivacaine gel samples were spread as thin layers on the inside walls of two quartz UV
cells. The cells were exposed uncovered, sample side up, to UV (254 and 365 nm)
(output: 300mW/cm2 at 6”) and white light under ambient conditions for 48 hrs. After 48
hrs, the light source was removed and sample was analyzed.
2.6.5. Oxidative degradation
Hydrogen peroxide (3%, 3mL) was added to diluted mepivacaine gel sample. This
sample was then analyzed after 4 h to estimate degradation of mepivacaine.
2.6.6. Acid degradation
Hydrochloric acid (5N, 3mL) was added to gel sample and heated at 60◦C for 4 h. After 4
h, the solution was brought to ambient temperature and to this solution, 6mL of
acetonitrile was added and drug was extracted.
2.6.7. Base degradation
NaOH (5N, 3mL) was added to gel sample and heated at 60◦C for 4 h. After 4 h this
solution was brought to ambient temperature and to this solution, 6mL of acetonitrile was
added and drug was extracted. Results of the test are as shown in Figs 2.35-2.39.
Fig 2.36: Chromatogram of
Mepivacaine exposed to U.V. Light for
48hrs.
Fig 2.35: Chromatogram of Reference
Mepivacaine
Chapter 2: Preformulation Studies
99
Results and Discussion
The stability and degradation profiles of mepivacaine in gel samples were evaluated with
the long-term objective of developing stable topical formulations of this drug.
Chromatogram of mepivacaine extracted in the gel sample at 210 nm showed no
interferences between the drug and excipient peaks. The stability profile of mepivacaine
gel samples at high temperature and light conditions was monitored by high performance
liquid chromatography (HPLC). Forced degradation of mepivacaine gel sample was
performed at extreme heat, light, acidic (pH<2.0) and alkaline (pH>10.0) conditions and
by addition of hydrogen peroxide (oxidizing agent). Mepivacaine was very stable to acid
and base treatment, no degradation product was seen. Also, it was found stable to
oxidation. There were no significant changes in the chromatograms obtained with
mepivacaine gels subjected to oxidative, heat, light, acid or base stress, when compared
to the chromatograms obtained with the reference sample. The physical appearance of the
gel, however, changed when it was subjected to heat stress—the gel appeared to lose its
viscosity.
The developed HPLC procedure separated the excipients and other peaks from the
mepivacaine peak and will be utilized for the analysis of mepivacaine in compounded
mepivacaine topical formulations.
Fig 2.39. Chromatogram of
Mepivacaine after acid treatment.
Fig. 2.40. Chromatogram of
Mepivacaine after alkaline treatment.
Fig 2.37: Chromatogram on thermal
degradation.
Fig 2.38: Chromatogram of
Mepivacaine on oxidation