Section (i): Brief account of...

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Section (i): Brief account of Pericalcitol

Pericalcitol (PCL) is the analog of 1,25-dihydroxyergocalciferol, the active form of vitamin

D2. Chemically, it is 19-nor-1,25-(OH)2-vitamin D2 or 19-nor-1,25-dihydroxyvitamin D2, with

the empirical formula of C27H44O3, which corresponds to a molecular weight of 416.64. PCL

is chemically designated as 19nor-1α,3β,25-trihydroxy-9,10-secoergosta-5(Z),7(E),22(E)-

triene and has the following structural formula:

PCL is a white, crystalline powder. PCL is marketed by Abbott Laboratories under the trade

name Zemplar. Zemplar Capsules (paricalcitol) are indicated for the prevention and treatment

of secondary hyperparathyroidism associated with chronic kidney disease (CKD) Stage 3 and

4, and CKD Stage 5 patients on hemodialysis (HD) or peritoneal dialysis (PD). Like 1,25-

dihydroxyergocalciferol, PCL acts as an agonist for the vitamin D receptor and thus lowers

the blood parathyroid hormone level.

PCL, the active ingredient in Zemplar Capsules, is a synthetically manufactured analog of

calcitriol, the metabolically active form of vitamin D. Zemplar is available as soft gelatin

capsules for oral administration containing 1 µg, 2 µg or 4 µg of PCL.

http://en.wikipedia.org/wiki/Analog_(chemistry)

http://en.wikipedia.org/wiki/1,25-dihydroxyergocalciferol

http://www.rxlist.com/script/main/art.asp?articlekey=11433

http://www.rxlist.com/script/main/art.asp?articlekey=21111

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PCL drug substance and PCL injection drug product is official in US pharmacopeia

[1]. There is an article reported for stability indicating assay of PCL in injection dosage form

[2]. There are few assays reported in the literature for the determination of PCL determination

in biological fluids. An LC-radioreceptor assay [3] and HPLC-MS/MS [4] were applied in

pharmacokinetic studies. The former is applied to evaluate the effect of hemodialysis on the

pharmacokinetics of PCL; the latter evaluated the effect of omeprazole on the

pharmacokinetics of paricalcitol in healthy subjects.

However, there are no studies describing quantification methods for PCL and its

impurities in its pharmaceutical form, Zemplar® (paricalcitol) capsules.

There is no known impurity listed in PCL USP monograph. In PCL injection USP

Monograph, a total nine degradants are listed named as related compound A , B, C, D, E, F,

G,H and I. No structures are revealed for these listed impurities by USP. Out of nine

degradants, related compound A and related compound B are given as oxidative degradants

and remaining seven related compounds C, D, E ,F, G, H and I are given as acid degradants.

Reverse phase HPLC method available in USP for the related substances of PCL is useful for

injectable formulation as well as for drug substance. This method is found to be not suitable

for the related substances estimation in oil based capsule formulation due to placebo

interference of Cremophor EL peaks which are present in formulation. Sample solutions were

found to be turbid when the diluent (water : acetonitrile in 50 : 50 v/v ratio) suggested in USP

is used for oil based capsule formulation due to formation of emulsion. So far to our present

knowledge, no validated stability indicating HPLC method for the determination of related

substances of PCL in oil based hard gelatin capsule formulation is available in literature.

This prompted the author to develop stability indicating HPLC methods for the assay

and impurities of PCL in oil based capsules formulation. This chapter describes development

and validation of a stability indicating methods for assay and impurities.

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Section (iii): Stability Indicating HPLC method for impurities of Paricalcitol

Capsules.

This section reports the various aspects relating to the development and

validation of stability indicating HPLC method for impurities in Paricalcitol (PCL)

capsule dosage form.

1. Experimental

1.1. Chemicals

PCL capsules are formulated in Dr Reddy’s laboratories Ltd, Hyderabad, India.

The reference standard of PCL is supplied by USP and standard is supplied by Dr.

Reddy’s laboratories limited, Hyderabad, India. The HPLC grade acetonitrile, ethanol,

dichloromethane, n-Hexane, isopropyl alcochol and analytical grade NaOH, HCl,30%

hydrogen peroxide are purchased from Merck, Darmstadt, Germany. High purity water

is prepared by using Milli Q Plus water purification system (Millipore, Milford, MA,

USA). The chemical names and structures of PCL is as shown below.

1.2. Determination of appropriate UV wavelength

The suitable wavelength for the determination of PCL and its impurities is

identified by taking the overlay spectra from 200–400 nm of all impurities and PCL

from PDA detector.

1.3. Instrumentation and chromatographic conditions

Waters HPLC System with a photo diode array detector is used for the

method development and force degradation studies. The HPLC system used for method

validation is waters HPLC system with diode array detector and Agilent 1100 series LC

system with variable wavelength detector (VWD). The data is monitored and processed

by using waters Empower Networking Software. The chromatographic column used is

an Inertsil Silica, 250mm x 4.6 mm column, with 3.0µ particle size. The

chromatographic condition follows a gradient program consisting of mixture of n-

Hexane, ethanol, and dichloromethane in the ratio of 900:30:30 v/v as mobile phase A

and of mixture of n-Hexane, ethanol, dichloromethane and isopropyl alcohol in the ratio

of 900:40:120:40 v/v as mobile phase B. The gradient program is : Time/% Mobile

phase B is 0.0/0, 18/0, 40/100, 70/100, 75/0, 85/0. The flow rate of the mobile phase is

1.5 ml min-1

. The column temperature is maintained at 30ºC and the detection

wavelength is 262 nm. The injection volume is 500µl.

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1.4. Diluent: n-Hexane is used as diluent.

1.5. Preparation of PCL diluted standard solution:

An accurately measured amount of 1ml of USP Paricalcitol RS ( each ml

contains about 508 µg of PCL per ml) is transferred into a 50 mL dry volumetric flask.

The volume made up with ethanol and mixed well to obtain a standard stock solution of

about 10 µg of PCL per ml. 2ml of the stock solution is then diluted to 50 ml with ethanol

and mixed well. 5ml of this solution is then diluted to 100 ml with n-hexane to obtain a

standard preparation of about 0.02 µg of PCL per ml. The specimen chromatogram of

diluent and PCL diluted standard solution is shown in fig.6.3.1.

1.6. Test Preparation for PCL pharmaceutical formulations:

30 capsules of PCL capsules (hardgelatin) are weighed (W1) and the contents are

emptied by cutting with a sharp blade and then squeezing each capsule without loss of

material into a 50 ml beaker. The empty shells are washed with dichloromethane and

dried at room temperature. The empty capsule shells are then weighed (W2). Average

weight of contents of capsules are calculated by subtracting W2 from W1. Then the

medicament, which is squeezed already from capsules, equivalent to 20 µg of PCL is

transferred into a 10 ml volumetric flask. The contents of the flask is then diluted up to

the volume with diluent and shaken vigorously for 5 minutes. The resultant clear solution

is used for injection. Placebo sample is prepared in the same way by taking the placebo

equivalent its weight present in a test preparation. The specimen chromatogram of

placebo and test samples is shown in fig.6.3.2 and 6.3.3.

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Fig 6.3.1: Specimen chromatograms of diluent and PCL diluted standard.

Fig 6.3.2: Specimen chromatogram of placebo for PCL capsules.

Diluent

Placebo

Diluted standard

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Fig 6.3.3: Specimen chromatogram of PCL capsules.

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1.7. Specificity:

Regulatory guidances in ICH Q2A, Q2B, Q3B and FDA 21 CFR section 211,

require the development and validation of stability-indicating impurities method for all

pharmaceutical dosage forms. However, the current guidance documents do not indicate

detailed degradation conditions in stress testing. The forced degradation conditions,

stress agent concentration and time of stress, are found to effect the % degradation.

Preferably not more than 20% is recommended for active materials to make the right

assessment of stability indicating nature of the chromatographic methods. Optimisation

of such stress conditions which can yield not more than 20% degradation is based on

experimental study. Chromatographic runs of placebo solution and samples subjected to

force degradation are performed in order to provide an indication of the stability

indicating properties and specificity of the method. The stress conditions employed are

acid, base, neutral and oxidant media, moisture, heat and light. After the degradation

treatments are completed, the samples are allowed to equilibrate to room temperature,

neutralized with acid or base (as necessary), and diluted with diluent to get the working

concentrations equivalent to test preparation. The samples are analyzed against a freshly

prepared control sample (with no degradation treatment) and evaluated for peak purity

by using photo diode array detector. Specific conditions are described below.

1.7.1. Placebo (excipients) interference:

Placebo solutions are prepared by taking the weight of placebo approximately

equivalent to its weight in the sample as described in the test preparation for PCL

capsules dosage form.

1.7.2. Effect of acid hydrolysis

Medicament equivalent to 20µg is treated with 0.2 ml of 0.5N ethanolic HCl and 2

ml of diluent. The solution is heated at 60°C for 1 hour. The resulting solution is diluted

to 10 ml with diluent to obtain a solution having final concentration of drug at about 2

µg ml-1

.

1.7.3. Effect of base hydrolysis

Medicament equivalent to 20µg is treated with 0.5 ml of 0.5N ethanolic NaOH

and 2 ml of diluent. The solution is heated at 60°C for 15 hours. The resulting solution is

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diluted to 10 ml with diluent to obtain a solution having final concentration of drug at

about 2 µg ml-1

.

1.7.4. Effect of oxidation

Medicament equivalent to 20µg is treated with 0.2 ml of 6% H2O2 in ethanol and 2

ml of diluent. The solution is heated at 60°C for 3 hours. The resulting solution is diluted

to 10 ml with diluent to obtain a solution having final concentration of drug at about 2

µg ml-1

.

1.7.5. Effect of moisture and heat

To evaluate the effect of moisture and heat, PCL medicament is distributed as thin

layer over two glass plates. One plate is then exposed to 25ºC/90% relative humidity for

7 days. Similarly another plate is exposed in an oven at 60ºC for 48 hours. Then, both

the samples are subjected to sample preparation using diluents as described in test

preparation.

1.7.6. Effect of UV and visible light

To study the photochemical stability of the drug product, Paricalcitol medicament

is exposed to 1200 K Lux of visible light and 200 W h/ m2 of UV light by using photo

stability chamber. After exposure the samples are subjected to sample preparation using

diluents as described in test preparation.

1.8. Method validation

1.8.1. Identification of RRT of potential degradants :

The Relative retention times(RRT’s) of 7 specified degradant impurities are summarized

in table 5.3.1.

Table 5.3.1. RRT of PCL specified degradants in new method.

S.No Name of the impurity RRT

1 Specified degradant 1 0.22






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Method validation is performed using stressed samples and or with PCL due to lack of

availability of standards for the specified degradants.

1.8.2. Precision

Precision (intra-day precision) of the impurities method is evaluated by using the

neutralised acid stressed sample as it contains 6 specified degradants except specified

degradant 6. The acid stressed sample is injected six times into the developed

chromatographic conditions described above. % of impurities are calculated against a

qualified PCL standard. RSD is then calculated for % of impurities individually obtained

for injections. The inter-day precision is also evaluated by six different samples by

spiking PCL on placebo matrix at 1% level. The intermediate precision (inter day

precision) of the method is also evaluated using different HPLC system and different

HPLC column on different day in the same laboratory.

1.8.3. Limits of Detection (LOD) and Limit of Quantification (LOQ)

The LOD and LOQ for PCL are determined at a signal-to-noise ratio of 3:1 and

10:1, respectively, by injecting a series of dilute solutions with known concentrations.

LOD and LOQ of each individual specified degradants are not estimated due to lack of

impurity standards. Precision study is also carried out at the LOQ level by injecting six

individual preparations of PCL spiked on placebo and % RSD is calculated.

1.8.4. Linearity

Linearity for PCL diluted standard is estimated by diluting stock solutions to the

required concentrations. The solutions are prepared at different concentration levels from

LOQ to 200% of the specification concentration level (1%) for PCL. The solutions are

then injected into the chromatographic conditions developed. The data of peak area

versus concentration is subjected to least-square regression analysis.

1.8.5. Accuracy

A study of recovery of PCL from placebo is conducted. Samples are prepared by

spiking PCL on placebo at different spike levels starting from LOQ to 150% of the

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specification level(1%). Sample solutions are prepared in triplicate for each spike level

as described in the test preparation and injected into the chromatographic conditions

developed. The % recovery is then calculated against PCL diluted standard and

compared against the known amounts spiked.

1.8.6. Robustness

To determine the robustness of the developed method, experimental conditions

are deliberately altered and the elution patterns, resolution between PCL and its

impurities are evaluated. Stressed sample from acid degradation and oxidation

degradation is mixed for robustness evaluation purpose as together they produce all the

specified degradants. The effect of flow rate is studied at 1.5 ± 0.2 ml min-1.

The effect of

the column temperature is studied at 30ºC ± 3ºC. The effect of the percent organic

strength for both mobile phase A and mobile phase B is studied by varying by −10 to

+10% while other mobile phase components are held constant.

1.8.7. Solution stability and mobile phase stability

The stability of PCL and its impurities in solution for the impurities method is

determined by leaving test preparation of PCL capsules in a tightly capped volumetric

flask at room temperature on bench top and by measuring the amounts of impurities at

different intervals. The stability of mobile phase is also determined by analysing freshly

prepared solution of PCL capsules for 5 days by using the same mobile phase during the

study period.

2. Results and discussion

2.1. Determination of suitable wavelength

The UV spectrum of PCL and all its known impurities were extracted in PDA

detector from 200-400 nm and is illustrated in fig.6.3.4. The spectrum shows three

absorption maxima which can give a good sensitivity for the all impurities of PCL. The

third absorption maxima observed at 262 is found to be optimum as at other two

maxima, placebo is having interference for 0.22, 0.25 and 0.26 RRT degradants.

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Fig 6.3.4: UV Spectra of Paricalcitol and its impurities.

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2.2. Optimization of chromatographic conditions

The HPLC procedure is optimized with a view to develop a stability indicating

impurities method for estimation of impurities of PCL in oil based capsules formulation. PCL

related substances method is available in USP for PCL Injection, which is having the nine

degradants. Out of nine degradants, 7 are due to the acid stress (5 major and 2 minor) and

two degradants due to peroxide stress. RRT has been captured for all nine degradants in USP

method. However, no chemical names, structures are given in USP for these degradants. No

standards are supplied by USP for these impurities. In the absence of chemical names and

structures, it was not possible to get the impurities standards from market. To develop the

impurities method for PCL oil based formulation, where in medicament liquid is filled into

hard gelatin capsules and sealed, initially attempts were made by using reverse phase

chromatographic conditions of the USP PCL injection method. PCL capsules formulations

are of very low dose, namely 1 µg, 2 µg and 4 µg per capsule. Due to very low concentration

of the drug present in the medicament, it is observed that a very high injection volume is

necessary. A minimum of 500µl injection volume is selected to get adequate method

sensitivity. Preliminary experiments are conducted to dissolve the excipients (mainly

medium chain triglycerides and cremophore EL) in acetonitrile. When placebo and

medicaments are dissolved in acetonitrile, it is observed that all placebo components are not

going into solution and some of excipients are found to be immiscible. The recovery of PCL

analyte is found satisfactory in acetonitrile, but due to presence of cremophore EL in

formulation, interference is observed at the retention time of PCL degradants. The

inconsistency of peaks is also observed due to excipient immiscibility in acetonitrile. Several

experiments are conducted to separate out the excipients peaks from degradants by

modifying the USP method gradient program and by changing the column, but without much

success, due to inconsistency of placebo peaks. Attempts are made in different combination

of other solvents like DMF, DMSO to selectively extract PCL from formulation, but studies

are not successful, as excipient peaks were still appearing at the RT of PCL and its

degradants in reverse phase method in all of the diluents. Studies revealed that the excipient

majorly responsible for interference is cremophor EL. Therefore, studies are conducted based

on the reported literature method to remove the cremophor EL from formulation by using

precipitation technique involving mercuric chloride in a reaction with cremophor EL to form

an insoluble complex in ethanol. The efforts made to

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remove the cremophore EL from formulation based on above mentioned literature

method [5] were successful, but PCL is getting degraded during sample preparation.

Based on the above studies, it is felt necessary to change the chromatographic

method from reverse phase to normal phase. This is to resolve the main constraint of

excipient solubility and immiscibility with most of the polar solvents, as normal phase

chromatography allows use non polar solvents as diluents, where in medium chain

triglycerides and cremophore EL are completely soluble. Additionally, due to polarity

reversal, interference due to cremophore EL can be easily avoided, as it elutes early in

normal phase due to its non polar nature.

Studies are conducted using 250 x 4.6mm, silica column by changing the

selectivity of the mobile phase to achieve symmetrical peak shapes and adequate

separation of all degradants from excipient peaks. It is noticed that isocratic elution

methods are not suitable separate the degradant peaks. Therefore, experiments were

conducted with different combination of solvents as mobile phase A and mobile phase

B and also by changing different gradient programmes.

Finally a mixture of n-Hexane: ethanol: dichloromethane (In ratio of 90: 3: 3

v/v) as mobile phase A and mixture of n-Hexane: ethanol: dichloromethane: isopropyl

Alcohol in the ratio of 90 : 4 : 12 : 4 v/v as mobile phase B are found suitable with

adequate separation of all degradant peaks and avoiding interference. The optimum

gradient program (T/%B) is set as 0/0, 18/0, 40/100, 70/100, 75/0, 85/0.

Identification of RRT’s of degradants in the new method:

After the success in resolving the issue of excipient interference, a challenge

faced is to identify the RRT’s of the major degradants in the new normal phase method,

as the structures and standards are not available. Therefore, it is decided to collect the

fractions of all major degradation products in USP method and inject in newly

developed normal phase method to identify the RRT’s of the major degradants. PCL is

subjected to stress with peroxide and acid as per USP PCL injection related substance

method as given below.

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Acid degradation solution 1 – for getting unknown related compound D, F, G and H

1.0 mL of USP Paricalcitol solution RS is taken into 10 ml volumetric flask and

1 mL of 0.1N HCl is added. The resultant solution is heated at 70°C for 2 hrs and

diluted to volume with diluent (acetonitrile : water in 50 : 50 ratio) and mixed.

Acid degradation solution 2 – for getting unknown related compound C.

0.5 ml of USP Paricalcitol solution RS is taken into 10 ml volumetric flask and

0.5 ml of 0.5N ethanolic HCl is added. The resultant solution is heated at 60°C for 90

minutes and diluted to volume with diluent ( acetonitrile : water in 50 : 50 ratio) and

mixed.

Oxidative degradation solution – for getting unknown related compound A and B

2.0 mg of PCL standard is accurately weighed and transferred into 5 ml

volumetric flask. 0.5 ml of ethanol and 0.5 mL of 50 % Hydrogen Peroxide solution is

added. The resultant solution is kept on bench top for 1 hour and diluted to volume with

diluent (50 % acetonitrile and 50 %water) and mixed.

A 500µL of injection volume of acid degradant solution 1, acid degradant

solution 2 and oxidative degradant solution are injected in PCL Injection USP method

using (Alltima C18, 250 x 4.6 mm, 5µm) as main column and (Altima C18, 75 x 4.6

mm, 5µm) as guard column with mobile phase containing a gradient mixture of solvent

A and B. A mixture of Milli Q Water and acetonitrile is used in 85:15 v/v as solvent A

and acetonitrile is used as solvent B. The gradient program is set as (T/%B) 0/35, 25/95,

45/95, 47/35, 60/35. The eluted compounds are monitored at 262nm.

Fractions of all major degradants (A,B,C,D,F,G,H) are collected based on the

RRT’s given in USP. As per USP PCL Injection method, unknown related compound

“E and I” forms in very less concentration. These compounds are not formed with the

specified degradation conditions and hence no fractions collected for these peaks. All

fractions of impurities are pooled and dried by hot air and reconstituted with

dichloromethane and then injected into newly developed normal phase method. Based

on the injections, RRT’S of the major degradants are identified in the in new method.

RRT comparison in USP PCL injection method and newly developed PCL capsule

method is summarized in table 6.3.2.

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Table 6.3.2. Comparative RRT’s of degradants in USP and New method

Source Name RRT as per USP

Injection reverse

phase method

RRT as per

capsule normal

phase method

Oxidative

degradation

Unknown Related

compound-A

0.63 1.16

Oxidative

degradation

Unknown Related

compound-B

0.79 1.12

Acid degradation 2 Unknown Related

compound-C

0.89 1.05


compound-D

0.95 1.16


compound-E

1.32

( not forming)

Not applicable


compound-F

1.57 0.64


compound-G

1.66 0.64


compound-H

1.74 0.67


compound-I

1.79

(not forming)

Not Applicable

Thermal

degradation

Specified

degradant-1

Not Detected* 0.22

Thermal

degradation

Specified

degradant-2

Not Detected* 0.25

Thermal

degradation

Specified

degradant-3

Not Detected* 0.26

(* Not detected due to non-polar nature and also due to interference from cremophor EL)

In the new method of PCL Capsules, 3 new thermal degradants are also observed

in the PCL capsule formulation samples which are not possible to see in the USP

method. However, unknown compound A is co eluting with the unknown compound D

(1.16 RRT) and unknown compound F is co eluting with unknown compound G (0.64

RRT). Efforts made to separate them are not successful due to limitations of normal

phase. On the basis of the degradation study and stability data in capsule formulation, it

is observed that only four acid / oxidative degradants ( at RTT 0.64, RRT 0.67, RRT

1.05 and RRT 1.16 ) and 3 new thermal degradants (at RRT 0.22, RRT 0.25, RRT 0.26)

are present. Other degradants (like RRT 1.12) are not forming in capsule formulations

upon manufacturing and upon stability and hence not part of this present method study

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further. The 3 new thermal degradants along with acid and oxidation degradants are

designated in the new method as given in table 6.3.3.

Table 6.3.3. RRT’s of specified degradants in new normal phase method.

S.No Name of the impurity RRT Nature of the Impurity

1 Specified degradant 1 0.22 Thermal degradant



4 Specified degradant 4 0.64 Acid degradant



7 Specified degradant 7 1.16 Oxidation degradant

The fractions of all the unknown impurities listed in USP are collected and subjected

to mass spectroscopic study to understand the possible structures. The data is

summarized in table 6.3.4.

Table 6.3.4. HRMS data of specified degradants of PCL.

USP name Impurities

Name RRT

(about)

Observed m/z

by HRMS

Observed

molecular

formula

Exact

Mass

[M]

Exact

Molecular

Formula

NA Specified

degradant-1 0.22

381.3141

[M+H]+

C27H41O 380.3141 C27H40O

NA Specified

degradant-2 0.25

381.3118

[M+H]+

C27H41O 380.3118 C27H40O

NA Specified

degradant-3 0.26

381.3129

[M+H]+

C27H41O 380.3129 C27H40O

Unknown

compound F

Specified

degradant-4 0.64

381.3140

[M-18]+

C27H41O 398.3140 C27H42O2

Unknown

compound G

Specified

degradant-4 0.64

381.3138

[M-18]+

C27H41O 398.3138 C27H42O2

Unknown

compound H

Specified

degradant-5 0.67

399.3247

[M+H]+

C27H43O2 398.3247 C27H42O2

PCL Active 1.00

399.3256

[M-18]+

C27H43O2 416.3256 C27H44O3

Unknown

compound C

Specified

degradant-6 1.05

458.3638

[M+CH3CN]+

C29H48NO3 416.3638 C27H44O3

Unknown

compounds A

Specified

degradant-7 1.16

433.3313

[M+H]+

C27H45O4 432.3313 C27H44O4

Unknown

compounds D

Specified

degradant-7 1.16

417.3350

[M-18]+

C27H45O3 434.3350 C27H46O4

Unknown

compounds B

Specified

degradant-8 1.12

415.3229

[M-18]+

C27H43O3 432.3229 C27H44O4

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The probable structures are hypothesized as shown in figure 6.3.5. based on mass spectral

study and based on the chemistry of the PCL molecule and based on elution order in new

method.

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The HRMS spectra of all above impurities are depicted in figures 6.3.6 to 6.3. 16.

Figure 6.3.6. : HRMS spectra for specified degradant-1 at RRT 0.22:

Figure 6.3.7. HRMS spectra of specified degradant-2 at RRT 0.25.

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Figure 6.3.8. HRMS spectra of specified degradant-3 at RRT 0.26

Figure 6.3.9 : HRMS spectra of specified degradant-4 at RRT 0.64

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(USP unknown compound F):


(USP unknown compound G):


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(USP unknown compound H)

Figure 6.3.12. HRMS spectra of PCL.

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(USP unknown compound C)

Figure 6.3.14. HRMS spectra of specified degradant-7at RRT 1.16

(USP unknown compound A)

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Figure 6.3.15. HRMS spectra of specified degradant-7at RRT 1.16

(USP unknown compound D)


(USP unknown compound B)


2.3.1. Precision

The precision of test method (intra-day precision) is evaluated by injecting

the neutralized acid stressed sample. Acid stressed sample is injected six times into

the chromatographic condition developed. Relative standard deviations of % of

PCL and its impurities are evaluated. Intra-day precision is also demonstrated for

PCL spiked on placebo matrix at 1% level. Inter day Precision study is conducted

on a different day with different mobile phase, with different HPLC and different

column. Test preparations of sample are prepared similar to intra-day precision and

the Relative standard deviations of % of PCL and its impurities are evaluated. The

% RSD values are presented in table 6.3.5 and 6.3.6. % RSD values of less than

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15% for PCL and its impurities shows that method is precise and work

satisfactorily on different day, with different column and different HPLC system.

2.3.2. LOQ and LOD

The limit of detection, limit of quantification are determined by following signal

to noise ratio method for PCL diluted standard. A precision study also conducted at

LOQ level for PCL. The results shows that method is sensitive enough to quantify

impurities well below the ICH reporting threshold of 0.1%. The data is summarized in

table 6.3.7.

2.3.3. Linearity

A linear calibration plot for PCL diluted standard is drawn over the calibration

range LOQ to 150% of the specification level of 1.0%. Correlation co-efficient for

PCL is found to be greater than 0.997. The regression analysis results are shown in

table 6.3.8. The results indicates that an excellent correlation exists between the peak

area and concentration of the analyte for PCL. The linearity graphs are presented as

figure 6.3.17.

2.3.4. Accuracy

The percentage recovery of PCL in presence of placebo matrix of PCL capsules

from LOQ to 150% spike level are in the range of 98.8% TO 113.7%. The % recovery

values for PCL are presented in table 6.3.9. The data shows that the method is having

capability to estimate accurately in presence of placebo matrix of PCL capsules

Table 6.3.5: Results of Inter-day precision of test method for PCL and its impurities.

S.No

PCL % of Specified degradant

1 2 3 4 5 6 7

1 0.98 0.46 0.17 0.56 0.58 0.60 ND 0.91

2 0.94 0.47 0.18 0.54 0.59 0.59 ND 0.95

3 0.96 0.42 0.19 0.55 0.58 0.64 ND 0.92

4 0.95 0.48 0.18 0.57 0.60 0.63 ND 0.94

5 0.99 0.46 0.20 0.59 0.62 0.62 ND 0.96

6 0.93 0.50 0.21 0.58 0.63 0.65 ND 0.91

Avg 0.96 0.47 0.19 0.57 0.60 0.62 NA 0.93 %RSD 2.4 5.7 7.8 3.3 3.5 3.7 NA 2.3

361

Table 6.3.6: Results of intra-day precision of test method for PCL and its impurities.

Table 6.3.7: LOD , LOQ data of PCL

Name

LOD

in %

S/N

ratio

LOQ

in %

S/N

ratio

PCL 0.023 3.2 0.071 9.8

Precision at LOQ :

Name of

the Active Sample No. % PCL Mean %RSD

PCL

1 0.085

0.083 3.9

2 0.079

3 0.087

4 0.081

5 0.085

6 0.080

Recovery at LOQ :

Name of the

active

Sample

No.

‘µg PCL

added

‘µg’ PCL

found % Recovery

%

Mean

recovery

PCL

1 0.0016 0.0017 106.3

104.2 2 0.0016 0.0016 100.0

3 0.0016 0.0017 106.3

Sample

No.

PCL

% of specified degradants

1 2 3 4 5 6 7

1 1.01 0.51 0.22 0.45 0.62 0.52 ND 0.88

2 1.05 0.53 0.20 0.49 0.61 0.55 ND 0.92

3 1.09 0.50 0.18 0.50 0.63 0.54 ND 0.91

4 1.06 0.49 0.21 0.51 0.62 0.57 ND 0.93

5 1.04 0.51 0.22 0.53 0.64 0.56 ND 0.95

6 1.05 0.53 0.21 0.54 0.65 0.59 ND 0.92

Avg 1.05 0.51 0.21 0.50 0.63 0.56 NA 0.92 %RSD 2.5 3.1 7.3 6.4 2.3 4.4 NA 2.5

362

Fig.6.3.17: Linearity graph of Paricalcitol.

Table 6.3.8 . Recovery data of PCL from formulation matrix.

S.No Spike level

As % of test conc.

‘µg’ added ‘µg’ found

(recovered) % Recovery

1 50% 0.50% 0.0102 0.0116 113.7

107.8 2 50% 0.50% 0.0102 0.0110 107.8

3 50% 0.50% 0.0102 0.0104 102.0

1 75% 0.75% 0.0150 0.0160 106.7

104.5 2 75% 0.75% 0.0150 0.0154 102.7

3 75% 0.75% 0.0150 0.0156 104.0

1 100% 1.00% 0.0203 0.0214 105.4

105.7 2 100% 1.00% 0.0203 0.0219 107.9

3 100% 1.00% 0.0203 0.0211 103.9

1 125% 1.25% 0.0252 0.0249 98.8

101.2 2 125% 1.25% 0.0252 0.0258 102.4

3 125% 1.25% 0.0252 0.0258 102.4

1 150% 1.50% 0.0305 0.0325 106.6

105.6 2 150% 1.50% 0.0305 0.0323 105.9

3 150% 1.50% 0.0305 0.0318 104.3

y = 1022641.47725 x - 61.69

0

10000

20000

30000

40000

50000

0 0.01 0.02 0.03 0.04 0.05

pea

k a

rea

Concentration in µg/ml

Pericalcitol

R = 0.999

363

2.3.5. Robustness

To determine the robustness of the developed method, experimental conditions are

deliberately altered and the separation between PCL and its impurities and tailing factor for

PCL and its impurities are recorded. The flow rate of the mobile phase is 1.5 ml min-1

. To

study the effect of flow rate on the resolution, flow is changed by 0.2 units from 1.3 to 1.7

ml min-1

. The effect of the column temperature on separation is studied at 25⁰C and 35⁰C

instead of 30⁰C. The effect of the percent organic strength on separation is studied by

varying −10 to +10% while other mobile phase components are held constant.

In all the deliberately varied chromatographic conditions (flow rate, column

temperature and composition of organic solvent), all analytes are adequately resolved and

elution orders remained unchanged. RRT of all the known impurities for all deliberately

varied conditions along with original conditions are summarized in table 6.3.9. The

resolution between all critical pair components is greater than 2.0 and tailing factor for PCL

and its impurities is found to be less than 1.2.


The stability of PCL in diluted standard solution is estimated against freshly

prepared standard each time. The similarity factor for standard from initial to 5 days is

found to be within the acceptable limits. PCL test preparation prepared as per test method

is injected at Initial and at different intervals upto 5 days. The difference in % of

individual impurities and % of total impurities are found to be within the limits upto 5 days.

The results are summarized in table 6.3.10.

The stability of mobile phase-A & B on bench top is conducted for a period of

about 5 days. Paricalcitol test preparation prepared as per test method is injected into the

chromatographic conditions developed by preparing the solution freshly each time and by

using the stored mobile phase up to 5 days. The difference in % of individual impurities

and % of total impurities from initial to 5 days is found to be within the limits. The results

are summarized in table 6.3.11. From the above study it is established that the PCL diluted

364

standard is stable up to 2 days on bench top, test preparation of PCL capsules is stable upto

5 days on bench top and mobile phase is stable for a period of 5 days on bench top.

364

Table 6.3.9: Results of robustness study

parameter

RRT of Specified degradants

Spe.Deg 1 Spe.Deg 2 Spe.Deg 3 Spe.Deg 4 Spe.Deg 5 Spe.Deg 6 Spe.Deg 7

1.3 ml/min 0.264 0.294 0.309 0.642 0.673 1.084 1.169

1.5 ml/min 0.221 0.246 0.259 0.636 0.665 1.085 1.181

1.7 ml/min 0.209 0.232 0.244 0.617 0.645 1.071 1.147

27°C 0.23 0.257 0.271 0.637 0.666 1.074 1.152

30°C 0.221 0.246 0.259 0.636 0.665 1.085 1.181

33°C 0.229 0.255 0.268 0.633 0.673 1.075 1.157

M.P A ( ethanol ) 90% 0.27 0.313 0.33 0.69 0.714 1.082 1.172

M.P A ( ethanol )100% 0.221 0.246 0.259 0.636 0.665 1.085 1.181

M.P A ( ethanol )110% 0.234 0.257 0.271 0.622 0.649 1.053 1.113

M.P A (dichloromethane) 90% 0.239 0.268 0.282 0.647 0.674 1.081 1.175

M.P A (dichloromethane)100% 0.221 0.246 0.259 0.636 0.665 1.085 1.181

M.P A (dichloromethane)110% 0.232 0.26 0.273 0.649 0.675 1.085 1.179

M.P B ( ethanol ) 90% 0.226 0.253 0.266 0.63 0.66 1.08 1.165

M.P B ( ethanol )100% 0.221 0.246 0.259 0.636 0.665 1.085 1.181

M.P B ( ethanol )110% 0.258 0.286 0.302 0.645 0.672 1.072 1.137

M.P B (dichloromethane) 90% 0.222 0.244 0.257 0.637 0.662 1.077 1.162

M.P B (dichloromethane)100% 0.221 0.246 0.259 0.636 0.665 1.085 1.181

M.P B (dichloromethane)110% 0.232 0.259 0.273 0.642 0.67 1.074 1.152

M.P B (isopropanol) 90% 0.219 0.243 0.255 0.623 0.652 1.085 1.173

M.P B (isopropanol)100% 0.221 0.246 0.259 0.636 0.665 1.085 1.181

M.P B (isopropanol)110% 0.226 0.251 0.264 0.64 0.668 1.072 1.147

Spe.Deg = Specified degradant

365

Table 6.3.10. Results of solution stability

Duration

In days

% of the impurity of Specified degradants upon storage on bench top.

TI

1 2 3 4 5 6 7 MUI

0 0.0820 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.0820

1 0.0640 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.0640

3 0.0740 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.0740

5 0.0974 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.0974

Maximum

difference

from initial

0.02 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.02

MUI =Maximum unknown individual impurity, TI = Total impurities

Table 6.3.11.Results of mobile phase stability on bench top

Duration in

days

% of Specified degradants in Paricalcitol test preparation

TI

1 2 3 4 5 6 7 MUI

0 0.0820 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.0820

1 0.0633 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.0633

3 0.1123 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.1123

5 0.0829 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.0829

Max

difference

from initial

0.03 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.03

MUI =Maximum unknown individual impurity, TI = Total impurities

366

5.3.7. Results of specificity studies

All the placebo and stressed samples prepared are injected into the HPLC system

with photodiode array detector as per the described chromatographic conditions.

Chromatograms of placebo solutions have shown no peaks at the retention time of PCL and

its impurities. This indicates that the excipients used in the formulation do not interfere in

estimation of impurities in PCL capsules.

Degradation is not observed in light exposure or moisture. Significant degradation is

observed in acid, base, water, peroxide and thermal degradation. All degradant peaks are well

resolved from each other and from PCL peak in the chromatograms of all stressed samples.

The chromatograms of the stressed samples are evaluated for peak purity of PCL using

Waters Empower Networking software. For all forced degradation samples, the purity angle

(the weighted average of all spectral contrast angles calculated by comparing all spectra in the

integrated peak against the peak apex spectrum) is found to be less than threshold angle (the

sum of the purity noise angle and solvent angle, the purity noise angles across the integrated

peak) for PCL peak. The details of each impurity formed in each stressed sample is

summarised in table 6.3.12. The stressed samples are subjected to mass balance study by

assaying the samples for PCL content apart from estimating the total impurities. The sum of

total % of impurities and assay is presented as mass balance, which is found to be in the range

of 98.3 to 100.4% for the stressed samples. The data indicates that there is no co-elution of

any degradants in the PCL peak and no impurity which is missing, as the mass balance is

close to 100%. Thus, this method is considered as specific and "Stability indicating”. The

chromatogram and purity plots of all stressed samples are shown in figs 6.3.18 to 6.3.24.

367

Table 6.3.12 : Summary of forced degradation studies

Stress condition

% of Paricalcitol Specified degradants

MUI TI % Assay

Mass

balance

1 2 3 4 5 6 7

Control sample 0.082 ND ND ND ND ND ND ND 0.082 99.8 99.9

Acid stress 0.46 0.17 0.56 0.58 0.60 ND 0.91 0.09 3.40 96.4 99.8

Base stress 2.04 0.52 1.48 ND ND ND ND 0.50 4.65 95.0 99.7

H2O2 stress 0.11 ND 0.11 ND ND ND ND ND 0.26 99.8 100.1

Sunlight stress 0.08 ND ND ND ND ND ND ND 0.08 99.8 99.9

UV light stress 0.07 ND ND ND ND ND ND ND 0.07 100.2 100.3

Thermal stress 1.02 0.29 0.87 ND ND ND ND 0.32 2.55 97.2 99.8

Humidity stress 0.08 ND ND ND ND ND ND ND 0.08 99.7 99.8

TI: Total impurities; MUI = Maximum unknown impurity, ND : Not detected.

368

Fig 6.3.18: Chromatogram and purity plot of acid stressed PCL capsules..

369

Fig 6.3.19: Chromatogram and purity plot of base stressed PCL capsules.

370

Fig 6.3.20: Chromatogram and purity plot of H2O2 stressed PCL capsules.

371

Fig 6.3.21: Chromatogram and purity plot of sun light stressed PCL capsules.

372

Fig 6.3.22: Chromatogram and purity plot of UV light stressed PCL capsules.

373

Fig 6.3.23: Chromatogram and purity plot of heat stressed PCL capsules.

374

Fig 6.3.24: Chromatogram and purity plot of humidity stressed PCL capsules.

375

3. Conclusion:

A precise, sensitive, specific, accurate, validated and well-defined stability indicating

LC method for the determination of degradation products of PCL in oil based capsules

formulation is described. The behavior of PCL under various stress conditions is studied. All

of the degradation products are found to be well separated from each other except those which

are structurally closely related. Although reverse phase USP method gives best separation for

unknown degradants, it is found to be not suitable for oil based formulations. The new

method developed is found to be the best suitable for oil based PCL capsule formulation.

HRMS studies and the elution patterns in normal phase chromatography lend supporting

evidence to the structures proposed for unknown compounds which are declared as unknown

degradants in USP. 3 new thermal degradants are found due this study which are not declared

in literature till date and which are not possible to get detected in USP method. Although

further study is needed to confirm the structures by NMR, this study has given a way forward

approach for structural characterization of degradants of PCL. The information presented in

this study could be very useful for quality monitoring of oil based PCL pharmaceutical dosage

forms and can be used to check drug quality during stability studies.

376

Reference

1. USP Monograph for Paricalcitol and Paricalcitol Injection USP 32 sup 2

2. Raquel, Ortega., Natalia Navasi., J. AOAC Int..,2011;94(2):543-549.

3. Cato, A., Cady, W.W., Soltanek, C., Qasawa, B., Chang, M., Stoll, R., Am. J. Kidney

Dis., 1998; 32: S55–S60

4. Palaparthy, R., Pradhan, R.S., Chan, J., Rieser, M., Chira, T., Galitz, L., Awni, W.,

Williams, L.A., Biopharm. Drug Dispos., 2007; 28: 65–71

5. Perdue, James, D., Seaton, Pamela, J., Tyrell, John, A., DeVido, Daniel, R., J.

Pharm.Biomed.Anal., 2006;41(1):117-123

323

Section (V): Stability Indicating HPLC Assay method for Paricalcitol capsules.

This section reports the various aspects relating to the development and validation of

stability indicating HPLC method for assay of Paricalcitol (PCL) capsules.

1. Experimental

1.1. Chemicals

Samples of PCL API are received from M/s. Formosa laboratories, Taiwan. PCL

Capsules of 1,2 and 4 µg & all excipients are received from formulation R&D, Dr Reddy’s

Laboratories, Hyderabad, India. The HPLC grade dichloromethane, n-Hexane, isopropyl

alcohol, methanol and analytical grade NaOH, HCl, 30% hydrogen peroxide are purchased

from Merck, Darmstadt, Germany. High purity water is prepared by using Millipore Milli-Q

plus purification system.

1.2. Determination of appropriate UV wavelength

The suitable wavelength for the determination of PCL in diluent is identified by scanning

over the range 200–400 nm with a Shimadzu UV-160 (Shimadzu, Japan) double beam

spectrophotometer.

1.3. Instrumentation and chromatographic conditions

The Waters HPLC System with a photo diode array detector is used for the

method development and force degradation studies .The data is monitored and processed

using Waters Empower Networking software. The HPLC system used for method validation

is waters HPLC system with diode array detector and Agilent 1100 series LC system with

variable wavelength detector (VWD). The chromatographic column used is Lichrosphere

Silica 60 , 250 x 4.0 mm, 5µm. The chromatographic condition follows an isocratic elution

with a mobile phase consisting of mixture of n-Hexane, dichloromethane, isopropyl alcohol

and methanol in the ratio of 800:200:120:50 v/v respectively. The flow rate of the mobile

phase is 1.0 ml min-1

. The column temperature is maintained at 40ºC and the detection

wavelength is 252 nm. The injection volume is 400µl.

1.4. Diluent: Dichloromethane is used as diluent.

324

1.5. Preparation of standard drug solution:

The stock solution of PCL USP reference standard (equivalent to about 10 µg. ml-1

of PCL) is prepared in diluent. The working standard solution (0.2 µg ml-1

of PCL) is

obtained by dilution of the stock solution in diluents for sample analysis purpose. The

Specimen chromatograms of diluent and standard are shown in fig.6.2.1.

Fig 6.2.1: Specimen chromatogram of diluent and PCL standard 0.2µg ml-1

.

1.6. Test Preparation for pharmaceutical formulations:

10 capsules of PCL capsules (hardgelatin) are taken and the contents are emptied by

cutting with a sharp blade and then squeezing each capsule without loss of material into a 50

ml beaker. The blade is washed with about 15 ml of diluent into the beaker, contents of the

beaker is then swirled to dissolve in the diluent and mixed. The resultant clear solution is then

Blank

Standard

325

transferred through funnel in to a volumetric flask. (50 ml for 1µg capsules, 100 ml for 2 µg

capsules and 200 ml for 4 µg capsules). The empty shells are then sonicated with adequate

amount of diluent for about 5 minutes and the solvent is then transferred in to the volumetric

flask. The empty shells are washed minimum 3 times with diluent and washings are

transferred in to the same volumetric flask. The contents of the flask is then diluted up to the

volume with diluent and mixed well. The resultant clear solution is used for injection. Placebo

sample is prepared in the same way by taking the placebo equivalent its weight present in a

test preparation. The specimen chromatogram of placebo and test samples is shown in

fig.6.2.2.

Fig 6.2.2: Specimen chromatogram of placebo and PCL Capsules.

1.7. Specificity:

Regulatory guidances in ICH Q2A, Q2B, Q3B and FDA 21 CFR section 211, require the

development and validation of stability-indicating potency of assays. However, the current

guidance documents do not indicate detailed degradation conditions in stress testing. The

forced degradation conditions, stress agent concentration and time of stress, are found to

Placebo

Test sample

326

effect the % degradation. Preferably not more than 20% is recommended for active materials

to make the right assessment of stability indicating nature of the chromatographic methods.

The discovery of such stress conditions which can yield not more than 20% degradation is

based on experimental studies. Chromatographic runs of placebo solution and samples

subjected to force degradation are performed in order to provide an indication of the stability

indicating properties and specificity of the method. The stress conditions employed are acid,

base, neutral and oxidant media, moisture, heat and light. After the degradation treatments are

completed, the samples are allowed to equilibrate to room temperature, neutralized with acid

or base (as necessary), and diluted with diluent to get the working concentrations equivalent

to test preparation. The samples are analyzed against a freshly prepared control sample (with

no degradation treatment) and evaluated for peak purity by using photo diode array detector.

Specific conditions are described below.

1.7.1. Placebo (excipients) interference:

Placebo solutions are prepared by taking the weight of placebo approximately

equivalent to its weight in the sample as described in the test preparation for PCL capsules

dosage form.

1.7.2. Effect of acid, base and peroxide :

PCL capsules medicament equivalent to 10 µg is treated with 1 ml of 0.1N methanolic

HCl, 1 ml of 0.1N methanolic NaOH and 0.1 ml of 6% H2O2 for 90 minutes, 17 hours and 2

hours respectively on bench top with continuous shaking. The resulting solutions are then

diluted to make 50 ml volume with diluents to get the final concentration of drug at about 0.2

µg ml-1

.

1.7.5. Effect of moisture and heat

To evaluate the effect of moisture and heat, PCL capsules medicament is distributed as

thin layer over two glass plates. One plate is then exposed to 25ºC/90% relative humidity for

7 days. Similarly another plate is exposed in an oven at 60ºC for 20 hours. Then, both the

samples are subjected to sample preparation using diluents as described in test preparation.

327

1.7.6. Effect of UV and visible light

To study the photochemical stability of the drug product, the PCL capsules medicament

is exposed to 1200 K Lux hours of visible light and 200 Watt hours/ m2 of UV light by using

photo stability chamber. After exposure the samples are subjected to sample preparation using

diluent as described in test preparation.


1.8.1. Precision

The precision (intra-day precision) of test method is evaluated by assaying the six

different individual preparation of the PCL Capsules 1µg, 2µg and 4µg respectively and

analyzed as per test method. The % assay is calculated against USP reference standard and the

relative standard deviation of six different assays of PCL is then calculated. The intermediate

precision (inter day precision) of the method is also evaluated using two different HPLC

systems and different HPLC columns in different days in the same laboratory.

1.8.2. Linearity

Linearity study for assay method is made using six different concentration levels in

the range of about 0.02- 0.4µg ml-1

of PCL (corresponding to 10 to 200% of assay of nominal

sample concentration of 0.2 µg ml-1

). The

data of peak area versus concentration is subjected

to least-square regression analysis.

1.8.3. Accuracy

A study of recovery of PCL from placebo matrix is conducted. Medicament equivalent

to about 1,2,5,7.5, 10, 12.5 and 15 µg is taken into 50 ml volumetric flask. The medicament

is then dissolved and diluted to obtain test preparation equivalent to about 10%, 20%, 50%,

75%, 100%, 125% and 150% of the target assay concentration (0.2 µg ml-1

) of the PCL

capsules. Sample solutions are prepared in triplicate for each spike level and assayed as per

test method. The % recovery is then calculated.

1.8.4. Robustness

To determine the robustness of the developed method, experimental conditions are

purposely altered one after the other to estimate their effect. Five replicate injections of

standard solution are injected under each parameter change. The effect of flow rate, pH,

column temperature and organic phase composition in mobile phase (dichloromethane,

isopropyl alcohol and methanol) on the tailing factor, theoretical plates of PCL peak and the

328

%RSD for peak areas of replicate injections of standard is studied at flow rates of 0.8 ml min-1

and 1.2 ml min-1

, at column temperatures of 35ºC and 45ºC and organic phase compositions

in mobile phase at + 10% respectively.


The solution stability of PCL in the assay method is carried out by leaving solutions of

both the test preparation and reference standard preparation in tightly capped volumetric

flasks at room temperature for 5 days. The same sample solutions is assayed periodically

during the study period.

The mobile phase stability is also carried out by assaying freshly prepared sample

solutions against freshly prepared reference standard solutions at periodic interval for 5days.

Mobile phase prepared is kept constant during the study period. The % RSD of assay of PCL

is calculated for the study period during mobile phase stability and solution stability

experiments.

2. Results and discussion

2.1. Determination of suitable wavelength

The UV spectrum of PCL recorded in the range 200-400 nm is illustrated in fig.6.2.3.

The spectrum indicates that 252 nm gives a good sensitivity for the assay.

Fig 6.2.3: UV Spectra of paricalcitol.

329

2.2. Optimization of chromatographic conditions

The HPLC procedure is optimized with a view to develop a stability

indicating assay method for estimation of PCL in oil based capsules formulation. PCL

assay method is available in USP for PCL Injection. To develop the assay method for PCL

based formulation, where in medicament liquid is filled into hard gelatin capsules and

sealed, initially attempts were made by using reverse phase chromatographic conditions of

the USP PCL injection method. PCL capsules formulations are of very low dose, namely

1 µg, 2 µg and 4 µg per capsule. Due to very low concentration of the drug present in the

medicament, it is observed that a very high injection volume is necessary. A minimum of

400µl injection volume is selected to get adequate method sensitivity. Preliminary

experiments are conducted to dissolve the excipients (mainly medium chain triglycerides

and Cremophore EL) in acetonitrile. When placebo and medicaments are dissolved in

acetonitrile, it is observed that all placebo components are not going into solution and

some of excipients are found to be immiscible. The recovery of PCL analyte is found

satisfactory in acetonitrile, but due to presence of Cremophore EL in formulation,

inconsistency of peaks is observed due to excipient immiscibility in acetonitrile. Several

experiments are conducted to separate out the excipients peaks from PCL by modifying

the USP method gradient program and by changing the column, but without much

success, due to inconsistency of placebo peaks. Studies revealed that the excipient majorly

responsible for interference is Cremophor EL. Therefore, studies were conducted based on

the reported literature method [5] to remove the Cremophor EL from formulation by using

precipitation technique involving mercuric chloride in a reaction with Cremophor EL to

form an insoluble complex in ethanol. The efforts made to remove the cremophore EL

from formulation based on above mentioned literature method are successful, but PCL is

getting degraded during sample preparation. Based on the above studies, it was felt

necessary to change the chromatographic method from reverse phase to Normal phase.

This is to resolve the main constraint of excipient solubility and immiscibility with most

of the polar solvents, as normal phase chromatography allows use non polar solvents as

diluents, where in medium chain triglycerides and Cremophore EL are completely soluble.

330

Additionally, due to polarity reversal, interference due to Cremophore EL can be easily

avoided, as it elutes early in normal phase due to its non polar nature.

Studies are conducted using 250 x 4.6mm, silica column by changing the

selectivity of the mobile phase to achieve symmetrical peak shapes and adequate

separation of all degradants and excipient peaks. It is noticed that isocratic elution

methods is suitable. Therefore, experiments are conducted with different combination of

solvents as mobile phase.

Finally a mixture of n-Hexane, dichloro methane, isopropyl alcohol and methanol in

the ratio of 800:200:120:50 v/v respectively as mobile phase was found suitable with

adequate separation of all degradant peaks and avoiding interference from placebo within a

run time of 17 minutes. Optimum flow rate and column temperatures are found be at 1.0 ml

min-1

and at 40ºC respectively.


2.3.1. Precision

Method repeatability (intra-day precision) is evaluated by assaying six samples,

prepared as described in the test preparation. The mean % assay values are found to be in the

range of 100.3 to 102.0 and % RSD for assay values are found to be in the range of 0.4 to 0.8

These are well with in the acceptance criteria i.e. mean % assay between 97.0 -103.0 and RSD

not more than 2.0 %. The intermediate precision (inter day precision) is performed by

assaying six samples on different HPLC systems and different HPLC columns in different

days as described in the sample preparation. The mean % assay values are found to be in the

range of 99.2 to 103.0 and % RSD for assay values are found to be in the range of 1.0 to 1.4.

The result shows good precision of the method (table 6.2.1 and 6.2.2).

331

Table 6.2.1: Results of precision (intra-day) of test method

Sample No.

Intra-day precision

Assay of PCL as % of labeled amount

For 1 µg capsules For 2 µg capsules For 4 µg capsules

1 102.0 100.5 101.8

2 100.3 101.2 101.4

3 102.0 101.4 101.3

4 100.5 100.8 100.5

5 101.8 101.1 102.0

6 100.4 100.4 101.0

Range 100.3 to 102.0 100.4 to 101.4 100.5 to 102.0

Mean 101.2 100.9 101.3

%RSD 0.8 0.4 0.5

Table 6.2.2: Results of precision (inter-day) of test method

Sample No.

Intra-day precision

Assay of PCL as % of labeled amount

For 1 µg capsules For 2 µg capsules For 4 µg capsules

1 102.1 101.3 101.8

2 101.6 101.8 99.2

3 99.5 101.4 100.9

4 100.1 102.1 103.0

5 100.6 101.1 102.6

6 101.3 99.2 102.9

Range 99.5-102.1 99.2-102.1 99.2-103.0

Mean 100.9 101.2 101.7

%RSD 1.0 1.0 1.4

2.3.2. LOQ and LOD

The LOQ and LOD are determined based on signal-to-noise ratios at analytical

responses of 10 and 3 times the background noise, respectively. The LOQ is found to be

0.01 μg ml -1

with a resultant %RSD of 4.6(n = 5). The LOD is found to be 0.003 μg ml.-1

2.3.3. Linearity

A linear calibration plot for assay of PCL is obtained over the calibration range 0.02-

0.4 µg ml-1

and the correlation co-efficient is found to be 1.000. The result shown in fig.6.2.4

indicates that an excellent correlation exists between the peak area and concentration of the

analyte.

332

Fig.6.2.4: Linearity of detector response graph

2.3.4. Accuracy

The percentage recovery of PCL in pharmaceutical dosage form of capsules is shown

in table 6.2.3 ranged from 97.4 to 100.9 and indicates high accuracy of the method.

Table 6.2.3: Recovery results of PCL.

Sample

No.

Spike

level

“µg/ml”

added

“µg/ml”

found

%

Recovery

Mean %

Recovery

1 10% 0.0203 0.0201 99.2

98.6 2 10% 0.0203 0.0201 99.2

3 10% 0.0203 0.0198 97.4

1 20% 0.0406 0.0404 99.5

99.4 2 20% 0.0406 0.0403 99.3

3 20% 0.0406 0.0404 99.5

1 50% 0.1016 0.1005 98.9

99.5 2 50% 0.1016 0.1011 99.5

3 50% 0.1016 0.1018 100.2

1 75% 0.1524 0.1519 99.7

100.1 2 75% 0.1524 0.1529 100.3

3 75% 0.1524 0.1528 100.3

1 100% 0.2032 0.2046 100.7

100.2 2 100% 0.2032 0.2027 99.8

3 100% 0.2032 0.2036 100.2

1 125% 0.2540 0.2540 100.0

100.2 2 125% 0.2540 0.2529 99.6

3 125% 0.2540 0.2563 100.9

1 150% 0.3048 0.3011 98.8

100.0 2 150% 0.3048 0.3073 100.8

3 150% 0.3048 0.3064 100.5

333

2.3.5. Robustness

In all the deliberately varied chromatographic conditions studied (flow rate, column

temperature, organic composition in mobile phase), the tailing factor, theoretical plates and

the % RSD for the PCL peak area for five replicate injections of standard is found to be

within the acceptable limit of not more than 2.0%, illustrating the robustness of the method

(table 6.2.4).

Table 6.2.4: Results of robustness study

Parameter

Observed value

Variation Tailing factor Theoretical

plates

%RSD

1.Flow rate 0.8 ml min-1 1.1 7326 1.5

1.0 ml min-1 1.1 6220 0.6

1.2 ml min-1 1.0 5371 0.9

2.Column temperature 35ºC 1.1 5447 0.3

40ºC 1.1 6220 0.6

45ºC 1.1 6253 0.3

3.Mobile phase (for

dichloromethane)

90% 1.1 5885 0.4

100% 1.1 6220 0.6

110% 1.1 5983 0.4

4. Mobile phase

(for IPA)

90% 1.1 7380 0.2

100% 1.1 7193 0.5

110% 1.1 6924 1.2

5. Mobile phase

(for methanol)

90% 1.1 7045 0.3

100% 1.1 6974 0.4

110% 1.1 7437 0.4


The difference in % assay of PCL test and standard preparations upon storage on

bench top is found to be less than 1.6% up to 5 days. Mobile phase stability experiments

showed that tailing factor and % RSD are less than 1.1and 0.5% respectively and theoretical

plates are more than 5914 up to 5days. The solution stability and mobile phase stability

experimental data confirms that standard and test preparations and mobile phase used during

assay determination are stable up to 5 days.

334

2.3.7. Results of specificity studies

All the placebo and stressed samples prepared are injected into the HPLC system

with photodiode array detector as per the described chromatographic

conditions.Chromatograms of placebo solutions have shown no peaks at the retention time of

PCL. This indicates that the excipients used in the formulation do not interfere in estimation

of PCL in capsules formulation dosage form.

Degradation is not found to be significant when exposed to light and humidity. In acid

hydrolysis, base hydrolysis, thermal stress and oxidative studies, significant degradation is

observed. All degradant peaks are well resolved from PCL peak in the chromatograms of all

stressed samples. The chromatograms of the stressed samples are evaluated for peak purity of

PCL using Waters Empower Networking software. For all forced degradation samples, the

purity angle (the weighted average of all spectral contrast angles calculated by comparing all

spectra in the integrated peak against the peak apex spectrum) is found to be less than

threshold angle (the sum of the purity noise angle and solvent angle, the purity noise angles

across the integrated peak) for PCL peak (table 6.2.5). This indicates that there is no

interference from degradants in quantitating the PCL in capsules dosage form. Thus, this

method is considered "Stability indicating”. The typical chromatogram and purity plots of all

stressed samples are shown in figs 6.2.5 to 6.2.11.

Table 6.2.5: Table of results of specificity

Stress condition % Assay Purity

Angle

Purity

Threshold

Stressed with 0.1 N methanolic HCl for about

90 min on bench top 96.2 2.437 5.235

Stressed with 0.1 N methanolic NaOH for

about 17 hours on bench top 98.4 0.857 1.083

Stressed with 6% H2O2 in methanol for about

2 hours on bench top 95.5 0.786 1.040

Exposed to Sunlight for 1.2 million Lux hours

in photo stability chamber 100.2 0.780 1.001

Exposed to UV light for 200watt hours/square

meter in photo stability chamber 100.3 0.778 0.967

Heated at 60° C for about 20 hrs in oven 98.3 0.801 1.072

Exposed to humidity at 25°C, 90% RH for

about 7 days 100.1 0.782 1.007

335

Fig 6.3.5: Chromatogram and purity plot of acid stressed PCL capsules.

Fig 6.3.6: Chromatogram and purity plot of base stressed PCL capsules.

336

Fig 6.3.7: Chromatogram and purity plot of H2O2 stressed PCL Capsules.

Fig 6.3.8: Chromatogram and purity plot of sun light stressed PCL capsules.

337

Fig 6.3.9: Chromatogram and purity plot of UV light stressed PCL capsules.

Fig 6.3.10: Chromatogram and purity plot of thermal stressed PCL capsules.

338

Fig 6.3.11: Chromatogram and purity plot of humidity stressed PCL capsules.

3. Conclusion:

A validated stability-indicating HPLC analytical method has been developed for the

determination of PCL oil based formulation in capsules dosage form. The results of stress

reveal that the method is selective and stability-indicating. The proposed method is simple,

accurate, precise, specific and has the ability to separate the drug from degradation products

and excipients of capsules dosage form. The method is suitable for the routine analysis of

PCL in either bulk powder or in other pharmaceutical dosage forms. The HPLC procedure can

be applied to the analysis of samples obtained during accelerated stability experiments to

predict the expiry dates of PCL in bulk and in formulations.

Section (i): Brief account of...

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