Venkata Narasimha Rao et al.

, Cogent Chemistry (2017), 3: 1275955

http://dx.doi.org/10.1080/23312009.2016.1275955

ANALYTICAL CHEMISTRY | RESEARCH ARTICLE

Ultra performance liquid chromatographic method
for simultaneous quantification of plerixafor and
related substances in an injection formulation
Received: 14 June 2016 G. Venkata Narasimha Rao1*, B. Ravi1, M. Sunil Kumar1, P. Manoj1 and R. Venkata Nadh2
Accepted: 20 December 2016
Abstract:Plerixafor (PLX) injections are administered to patients with cancers of lym-
*Corresponding author: G. Venkata
Narasimha Rao, Formulations Research phocytes (non-Hodgkins lymphoma) and plasma cells (multiple myeloma). The main
and Development Centre, Mylan
Laboratories Ltd, Jinnaram, Medak objective of the current study was to develop a short reverse phase chromatographic
502325, India method for the simultaneous quantification of PLX and its impurities, in an injection
E-mail: [email protected]
formulation, to reduce the time required for these quality tests. Furthermore, the
Reviewing editor: present work describes the role of nonalkyl branched nonquaternary ion pair reagent
Avninder Bhambra, De Montfort
University, UK in improving the peak shape and reducing column equilibration time. The separa-
Additional information is available at tion of PLX and its related substances is pH dependent (optimum pH=2.50) and was
the end of the article achieved on an octadecylsilyl (C18) column. The method was validated for its intend-
ed purpose in accordance with the current regulatory guidelines for validation. The
proposed method can be applied for quality control, release, and stability analyses of
active pharmaceutical ingredient, PLX, as well as finished products, PLX injections.

Subjects: Analytical Chemistry; Organic Chemistry; Applied & Industrial Chemistry

Keywords: plerixafor; potential impurities; UPLC; stability-indicating assay; validation

1. Introduction
Patients with non-Hodgkins lymphoma (NHL) and multiple myeloma (MM) require mobilization of
hematopoietic stem cells (HSCs) as a strategic treatment following high doses of chemotherapy

ABOUT THE AUTHORS PUBLIC INTEREST STATEMENT

G. Venkata Narasimha Rao received the Bachelor For a medicinal drug to be proven safe and
of Science (BSc), Master of Science (MSc-Applied effective, it must be tested for various quality
Chemistry) degrees from the Acharya Nagarjuna attributes before releasing into the market. Since
University of Guntur, Devi Ahilya University of most of the drugs are synthesized chemically and
Indore, India, respectively. During 2007-up to contain certain impurities which are clinically not
date, he is working as an analytical scientist with safe for human body. Different drug regulations
a designation of Associate Director, in Mylan require identification and quantification of these
Laboratories Ltd. He is presently perusing his impurities and control below a safety threshold
research in developing shorter chromatographic limits. The techniques associated with these
methods for various pharmaceutical drug estimations involve intensive testing and take time
products, especially cancer therapeutic drugs, by to release the product into the market and in the
exploring the supercritical fluid chromatography process eliminate lots of organic wastes.
and associated techniques. His research interests This research was aimed to develop new shorter
are aimed at reducing the analysis time and procedures of testing on the same technique for a
the solvent consumption for a better waste quick testing of the product at the quality control
management and safeguard of the environment. lab and release into the market. In this research,
Plerixafor a cancer drug can be tested in 9min for
critical quality attributes like amount of main drug
and impurities. As a result of reduced time, solvent
usage is also reduced.

2017 The Author(s). This open access article is distributed under a Creative Commons Attribution
(CC-BY) 4.0 license.

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(Montgomery & Cottler-Fox, 2007). Currently, mobilization is initiated using granulocyte colony stim-
ulating factor (G-CSF), which promotes the proliferation and differentiation of HSCs (Klocke,
Kuhlmann, Scobioala, Schabitz, & Nikol, 2008).

Several days of treatment are required with placebo+G-CSF for adequate mobilization (Devine
et al., 2008). Studies have shown that patients with NHL and MM exhibited a rapid increase (a 2.5-
fold increase compared with only G-CSF) in peripheral blood CD34+ cells after subcutaneous admin-
istration of Plerixafor (PLX, direct antagonist of CXCR4/SDF-1) at a dose of 240gkg1 (Hess et al.,
2007). PLX is an on-demand, well-tolerated HSC mobilizer with mild adverse effects (Calandra et al.,
2008; DiPersio, Micallef, et al., 2007; DiPersio, Stadtmauer, et al., 2007; Flomenberg et al., 2005).

PLX is a symmetrical bicyclam derivative with molecular formula C28H54N8 and molecular weight
502.78gmol1. The structural formula of PLX and its impurities are depicted in Figure 1. PLX is a
white to off-white, hygroscopic, crystalline solid. The PLX injection formulation is a sterile, preserva-
tive-free, clear, and colorless to pale yellow isotonic solution for subcutaneous injection. Each single-
use vial is filled to deliver 1.2mL of the sterile solution containing 24mg of PLX and 5.9mg of sodium
chloride in water. Mozobil is the brand name of the innovator (CHMP Assessment Report, 2009; Drug
Bank, 2010; Drugs at FDA, 2008; Mozobil, 2016; Mozobil, Genzyme Corporation, 2015).

PLX and its injection formulation are not official monographs in any of the pharmacopeia (USP, EP,
BP, JP, and IP). It has an orphan drug status, approved by the FDA in the USA and in the EU. Hence,
no official methods have been reported for the estimation of PLX and related substances. A litera-
ture survey revealed several publications regarding the pharmacodynamics, pharmacokinetics, and
therapeutic efficacy studies on PLX (DiPersio, Stadtmauer, et al., 2007; Gerlach, Skerlj, Bridger,
Schwartz, 2001; Hatse, Princen, Bridger, De Clercq, & Schols, 2002; Hendrix et al., 2000; Hbel et al.,
2004; Lack et al., 2005; MacFarland, Ewesuedo, Badel, & Calandra, 2007; Rosenkilde et al., 2004).

An HPLC method for the determination of PLX was reported by Mathrusri Annapurna, Sai Pavan
Kumar, Goutam, and Venkatesh (2012). The method uses an isocratic elution mode using tetra butyl
ammonium hydrogen sulfate (pH=3.37) and acetonitrile mixed in the ratio 58:42 (v/v). The runtime
was 10min for PLX, and no impurities were addressed in the method.

An HPLC determination method was reported by Reddy et al., for PLX and its impurities in drug
substance (Hanimi Reddy, Ravi Kumar, & Satyanarayana Murthy, 2015). In this method, three impu-
rities and PLX were determined in 24min using a gradient elution mode. The mobile phase was a
complex mixture with a binary composition. The mobile phase A contained perchloric acid
(1.0mL)+heptane sulfonic acid (5mM) (pH=2.0) (buffer) and acetonitrile in the ratio 80:20 (v/v)
and mobile phase B contained a mixture of buffer and acetonitrile in the ratio 20:80 (v/v). The formu-
lation was separated on a phenomenex Luna phenylhexyl (L11) 100mm4.6mm, 3m column.
The eluted components were detected at 210nm. The method used long-chain alkyl sulfonates in
the mobile phase, which required a longer equilibration time before analysis than do methods that
do not use long-chain alkyl sulfonates (Fanali, Haddad, Poole, Schoenmakers, & Lloyd, 2013;
Verpoorte & Baerheim, 1984).

Thus far, studies have been reported either on PLX determination or its impurities in active phar-
maceutical ingredients. Studies on the estimation of PLX and its impurities in presence of excipients
in an injection formulation were not available.

Thus, an attempt was made for developing an accurate reproducible method that uses a nonalkyl
branched, nonquaternary ion pair reagent as a buffer for improving the peak shape for PLX and its
impurities, in the presence of excipients.

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Figure 1. Chemical structures

of plerixafor and its potential
impurities.

2. Materials and methods

2.1. Chemicals and reagents

Sodium perchlorate monohydrate (ACS grade) was used to prepare the buffer and obtained from
Acros Organics. Perchloric acid (GR Grade), used for adjusting pH, was obtained from Merck special-
ties, India. Acetonitrile (HPLC grade) was used as a mobile phase component and was procured from
Rankem India. Milli Q water was used for preparing the mobile phase. A standard working solution
of PLX was prepared in house. Impurity standards were obtained internally. Small volumes of PLX
injection samples and placebo mixtures were prepared in the laboratory.

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2.2. Instrument and chromatographic conditions

The Integrated Acquity UPLC system used for the study was purchased from Waters Corporation,
Milford, USA and equipped with Waters photodiode array detector (PDA). Data collection and analy-
sis was performed using Empower software 2pro (Waters Corporation). The balance used for weigh-
ing the reference standards and samples was purchased from Metler Toledo. Separation was
achieved on a Waters acuity CSH C18 column with dimensions 50mm2.1mm I.D and a particle
size of 1.7m. A simple mobile phase consisting of sodium perchlorate buffer (0.02M, pH 2.5) and
acetonitrile (Mobile phase B) was pumped into the UPLC chromatograph using a gradient program
with varying compositions (v/v) of B, T/B% 0/12, 2/15, 5/15, 5.50/70, 6.50/70, 7/12, 8/12, at a flow rate
of 0.32mLmin1, with a column temperature of 35C throughout the run. Sample volume of 3 L was
injected into the chromatograph and detected at 210nm.

2.3. Preparation of standard and sample solution

A mixture of aqueous hydrochloric acid (0.01M) and methanol in the ratio 90:10 was used as a dilu-
ent for preparing the solutions of standard and samples (diluent).

2.3.1. Standard stock solution

A standard stock solution was prepared by dissolving 50mg of PLX working standard in 50mL of the
diluent.

2.3.1.1. Preparation of standard and sample solution for assay determination (0.1mgmL1). The
standard solution for assay of PLX was prepared by diluting the standard stock solution 10 times
(550mL) to obtain a concentration of 0.1mgmL1. The vials of PLX injection were pooled and 2mL
of the sample was diluted to 100mL and further diluted 520mL to obtain the 0.1mgmL1 concen-
tration, similar to that of the standard.

2.3.2. Preparation of standard solution for impurities determination (0.004mgmL1)

The standard solution for the determination of impurities was prepared by diluting 0.4mL of the
standard stock solution into 100mL with the diluent to obtain a concentration of 0.004mgmL1.

2.3.3. Preparation of sample and placebo solution for impurities determination

Sample solution was prepared by diluting 2mL of the pooled PLX injection to 20mL using the diluent
to obtain a concentration of 2mgmL1. Placebo equivalent to 2mL of the sample was taken and
diluted to 20mL with the diluent and mixed.

2.3.4. Preparation of spiked sample solution for impurities determination

Stock solution of all impurities was prepared by dissolving an appropriate quantity of each impurity
in the diluent to obtain a concentration of 1mgmL1. An appropriate volume of impurity stock solu-
tion was diluted with sample solution to get a final concentration of 0.5% for each impurity.

3. Method development and optimization

PLX has ionizable amino groups, and hence, the retention time of the drug is highly dependent on
the pH of the mobile phase. In the present study, the pH of the mobile phase was maintained acidic
(pH=2.5) by the addition of sodium perchlorate solution. The ion exchange interaction between
positively charged amino groups on PLX and negatively charged silanol groups on column resulted
in mixed-mode retention. Such a mixed-mode retention effect is eliminated by the addition of
acidic sodium perchlorate solution (0.1M), which causes ion suppression or maintains the silanol
groups in unionized form. This yields narrow and symmetrical peak.

Before initiating the development activity, information on impurities and their acceptable limits
was collected to define sample concentration and the range of the method. The maximum daily
dose of PLX is 40mg/day. Based on the daily dose, the qualification threshold did not exceed 0.5%,
and the identification threshold was 0.2%. Table 1 lists the chemical names and ICH limits for the
specified impurities. Method development was targeted to cover a range of 50150% of qualification

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Table 1. Chemical names and limits based on ICH for impurities

Name of the impurities Chemical names ICH* limits QT**
Impurity-1 (Hydroxy impurity) (4-((1,4,8,11-tetraazacyclotetradecan-1-yl)methyl)phenyl) NMT 0.50%
methanol
Impurity-2 (Methyl impurity) 1-[(4-methylphenyl)methyl]-1,4,8,11-tetraazacyclotetradec- NMT 0.50%
ane
Impurity-3 1,8-bis(4-((1,4,8,11-tetraazacyclotetradecane-1-yl)methyl) NMT 0.50%
benzyl)-1,4,8,11-tetraazacyclotetradecane
Impurity-4 1,11-bis(4-((1,4,8,11-tetraazacyclotetradecane-1-yl)methyl) NMT 0.50%
benzyl)-1,4,8,11-tetraazacyclotetradecane
*ICH The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use.
**QT Qualification Threshold based on maximum daily dose.

threshold for impurities and PLX for assay. A systematic approach was adopted for the method
development.

3.1. Selection of detection wavelength

The sensitivity of a method that uses a UV detector depends on the proper selection of wavelength.
An ideal wavelength is that which is maximally absorbed and provides an acceptable response for
the drug, which should not interfere with other peaks.

UV spectra of the drug and its impurities were recorded by scanning between 200 and 400nm.

The spectra of drug and its impurities were overlaid and the wavelength 210nm was selected
where the active analyte as well as impurities have sufficient response for detection and quantifica-
tion. The ultraviolet scans of PLX and the four potential impurities are depicted in Figure 2.

Based on the physicochemical properties of the drug substance and the solubility (freely soluble in
alcohols), the reverse phase chromatographic technique was selected for initial separation of the
drug from its impurities.

The first development trial was initiated with sodium perchlorate buffer (pH 2.5), Waters Acquity
HSS T3 (1002.1mm, 1.7m), column with linear gradient program using 100% Acetonitrile as the
second component. The pump was maintained at a constant flow rate of 0.35mL/min. The column
was maintained at 35C. Two microliter of spiked sample was injected into the chromatograph and
the peak responses were monitored. All impurities were resolved from main peak; however, separa-
tion among impurities was not achieved. Figure 3 illustrates the chromatogram obtained from trial 1.

Figure 2. Ultraviolet scan of

plerixafor and its impurities
between 200 and 400nm

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Figure 3. Chromatogram
obtained from trial 1.

Another attempt was made by changing the gradient program and keeping other parameters
unchanged. The patterns obtained from both trials did not differ significantly. The pattern obtained
for trial 2 is depicted in Figure 4.

The next trial was made by changing the column to Waters CSH C18, 502.1mm, 1.7m. The
remaining chromatographic parameters were unchanged. All impurities were appropriately sepa-
rated from each other. The resolution between impurity 1 and the PLX is further improved by modify-
ing the gradient program. The optimized chromatographic conditions are listed in section 2.1. A
specimen chromatogram obtained from the final method parameters is illustrated in Figure 5.

Figure 4. Chromatogram
obtained from trial 2.

Figure 5. Specimen
chromatogram from final
method for spiked sample.

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Table 2. Validation data of related substances of plerixafor

S. no Compound Retention USP USP No. of Linearity M.P# I.P# LOQ Accuracy LOD$ LOQ$
Venkata Narasimha Rao et al., Cogent Chemistry (2017), 3: 1275955

time resolution tailing theoretical (r2) %RSD %RSD %w/w 50% 100% 150% (%w/w) (%w/w)
(mins) (RT (Rs) (T) plates USP (%RSD) %w/w %w/w %w/w
ratio) tangent (%RSD) (%RSD) (%RSD)
method (N)
1 Impurity 1 0.837 (0.72) 1.14 8,074 0.998 1.67 1.50 87.5 (0.8) 91.1 (0.8) 93.6 (2.3) 87.7 (0.4) 0.0075 0.02421
2 Plerixafor 1.161 (1.00) 2.2 1.34 6,665 0.999 0.3 0.2 100.5 (1.5) 101.5 (0.8) 100.1 (0.9) 101.6 (0.7) 0.02 0.1
3 Impurity 2 3.000 (2.58) 10.8 1.20 5,655 0.997 1.22 1.15 88.3 (1.1) 106.4 (0.4) 99.4 (1.3) 100.0 (1.0) 0.0135 0.03216
4 Impurity 3 3.886 (3.35) 5.5 1.11 9,223 0.999 0.71 0.69 98.0 (0.9) 108.5 (0.5) 108.0 (0.8) 104.2 (0.2) 0.0150 0.02381
5 Impurity 4 4.459 (3.84) 3.2 1.40 7,717 0.997 0.57 0.50 101.6 (1.1) 89.2 (0.3) 86.8 (0.9) 87.9 (1.1) 0.0207 0.04600
#
Method precision (M.P), intermediate precision (I.P).
$
LOD, LOQ values are established by S/N ratio.

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Figure 6. Linearity graph for

impurities-1, 2, 3, 4 (related
substances) and plerixafor
(assay).

Table 3. Data on signal to noise ratio

Impurity name Concentration (%w/w) Signal (S) (V) S/N* ratio
Impurity 1 0.02421 1,024 12
Impurity 2 0.03216 654 10
Impurity 3 0.02381 724 9
Impurity 4 0.04600 816 10
*S/N signal to noise ratio.

4. Results and discussion

4.1. Method validation

The optimized method was fully validated for the assay of PLX and simultaneous determination of
impurities, as per the current ICH guidelines (Q2A (R1) validation of analytical procedures, 2005).

4.1.1. System suitability

System suitability parameters were measured to verify the system performance for the intended
analysis. Hence, system precision was determined on six replicate injections of standard

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preparations and the relative standard deviation (% RSD) was evaluated. In addition to the % RSD,
USP Resolution, USP tailing, and USP plate count were also evaluated and found to be satisfactory,
as per current USP requirements for a chromatographic peak (USP General Chapter<621>). The sys-
tem suitability results obtained are presented in Table 2.

4.1.2. Linearity
The linearity of the analytical method was tested to check its ability to elicit test results that are directly
proportional to the concentration of analyte in samples within a given range. Hence, different concen-
trations of individual impurities and standard working solution of PLX were prepared and injected into
the UPLC, and the chromatograms were recorded. The linearity of detector response was determined
by plotting a graph of peak areas versus concentrations. Plots of linearity experiments are illustrated in
Figure 6. The correlation coefficients, >0.997 for impurities and 0.999 for PLX, indicate that the method
satisfies a linear relationship between the concentration and peak response. The linearity range is be-
tween the limit of quantification (LOQ) and 150% of the sample concentration for all impurities and
between 50 and 150% of target sample concentration for assay determination. The linearity data for
all four impurities and PLX are listed in Table 2. The signal-to-noise ratio for each impurity standard
meets the criteria as per the ICH requirement and the data are presented in Table 3.

4.1.3. Precision
Precision of the test method was evaluated by injecting six individual samples (assay concentration)
and six individual samples spiked with all four impurities into the chromatograph. The % RSD values
from the six individual test preparations were found to be 0.3 for assay determination and below 2%
for all four impurities. The ruggedness (intermediate precision) of the method was determined using
another system and column for the analysis in a different day. The precision data are listed in Table
2. The data indicate that a low % RSD, concluding that the method is precise for assay and impurities
determination.

4.1.4. Accuracy
Accuracy of the analytical procedure expresses the degree of closeness of the obtained results with
true values. Samples for accuracy were prepared in triplicate by spiking PLX and impurities at differ-
ent levels in the placebo. The covered levels for assay were 50, 100, and 150% of target assay con-
centration (0.100mgmL1). The covered levels for impurities were LOQ, 50% (1mgmL1), 100%
(2mgmL1), and 150% (3mgmL1) of the sample concentration (2mgmL1). From the response of
the analyte peaks, the amounts recovered (in %) and % RSD were reported. Accuracy results are
summarized in Table 2.

The data indicate that the assay recovery results are between 100.1 and 101.5 with an RSD rang-
ing from 0.8 to 1.5% for all three samples. The % recovery of impurities lies in the range of 85110
with an RSD of between 0.3 and 2.3%. This indicates that the method is accurate and precise.

4.1.5. Specificity
The specificity of the method was determined by analyzing the diluent, standard solutions of PLX,
placebo, and the impurities spiked sample. The chromatograms of the diluent and placebos solu-
tions were evaluated for the interference of any peaks at the retention times of the analyte peaks.
No interference was found. The samples of PLX injection were subjected to stress conditions, chemi-
cal conditions such as, acid hydrolysis, base hydrolysis, and 3% of oxidant treatment, as well as
physical conditions, such as treatment with heat and light. A detailed forced degradation study has
been detailed.

4.1.5.1. Forced degradation study. Forced degradation studies were conducted on samples and on
the plain placebo to prove the specificity and stability-indicating power of the method. Specificity
was determined by exposing test solution to oxidation by hydrogen peroxide, acid hydrolysis, base
hydrolysis, heat and photolytic stress. A detailed procedure has been reported.

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Oxidation stress studies were provided by adding 1mL of 3% H2O2 to 2mL of the sample and stor-
ing for 48h on bench top. Acid hydrolysis was performed by adding 1mL of 0.5N HCl to 2mL of the
sample and storing for 24h on the bench top. Base hydrolysis was performed by adding 1mL of
0.5N NaOH and storing for 24h on the bench top. Heat stress was provided by exposing the sample
to 70C for 48h. Photolytic studies were carried as per the current ICH requirements i.e. by exposing
the sample to UV light (200 Watt h/m2), day light (1.2 million lux h) (Fanali et al., 2013). The stressed
samples were then further diluted to 20mL with the diluent and chromatographed as per the pro-
posed method. The percentage assay and peak purity of PLX peaks were evaluated using the PDA

Figure 7. Forced degradation

chromatograms of plerixafor.

(a) Peroxide degradation

(b) Thermal degradation

(c) Acid degradation

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Figure 7. (Continued)

(d) Base degradation

(e) UV degradation

(f) Light degradation

detector. The forced degradation chromatograms are illustrated in Figure 7. Table 4 presents the
results obtained from the stress studies.

The data in Table 4 indicate that the maximum degradation was observed in case of oxidation
(5%), followed by base hydrolysis (0.8%). The molecule appears to be extremely stable under light

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Table 4. Results of forced degradation studies

Stress conditions % Degradation Peak purity* Purity flag**
Impurity Purity Purity
angle threshold
Treated with 0.5N HCl (1ml) 0.2 Impurity-1 1.120 1.328 No
solution for 24h on bench top
Impurity-2 12.528 12.697
Impurity-4 1.595 1.800
Treated with 0.5N NaOH (1ml) 0.8 Impurity-1 2.327 2.693
solution for 24h on bench top
Impurity-2 9.083 11.358
Impurity-4 1.466 1.766
Treated with 3% H2O2 (1ml) solution 5.0 Impurity-1 0.384 0.408
for 48h on bench top
Impurity-2 5.558 8.645
Impurity-4 2.173 2.251
Treated with heat at 70C for 48h 0.2 Impurity-1 1.695 2.591
Impurity-2 6.581 7.965
Impurity-4 1.480 2.014
Exposed for sunlight 1.2million lux h 0.1 Impurity-1 9.059 9.470
Impurity-2 9.666 14.499
Impurity-4 1.940 2.312
Exposed for UV-light 200Whsq.m 1
0.1 Impurity-1 9.478 9.855
Impurity-2 8.427 9.989
Impurity-4 1.650 1.966
Note: If it is shows Yes then peak is non homogenous.
*Peak purity passes if purity angle<purity threshold.
**Purity flag: It indicates homogeneity of all peaks.

Table 5. Results of stability of standard solution and sample at room temperature

Stability interval (about) Plerixafor sample
Result Difference
Standard Sample-1 Sample-2 Sample-1 Sample-2
Initial NA 100.1 100.0 NA NA
24h 1.00 99.0 99.2 1.1 0.8

stress conditions. According to Waters Empower software, the peak is homogenous if the purity
angle is less than the purity threshold. The peak purity data indicated that all known impurities and
PLX peaks were homogenous and free from interference, and their estimation was unaffected in
presence of other degradant peaks. This confirms the stability-indicating power of the developed
method.

4.1.6. Solution stability and mobile phase stability

The solution stability of standard and samples was determined by storing both the test solutions of
sample and standard at room temperature for 24h. The similarity factor was determined for the two
standard solution responses, and %w/w concentrations were determined for assay of sample and
impurities. Tables 5 and 6 present the solution stability for assay and related substances,
respectively.

From Table 5, the similarity factor between the standard at zero hours and standard at 24h is
1.00. This indicates that the standard solution is stable up to 24h at room temperature.

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The difference of % concentrations of impurities obtained between sample at zero hours and that
obtained at 24h indicates that the sample solution is stable up to 72h.

4.1.7. Robustness
The robustness of an analytical method is a measure of its capacity to remain unaffected by small
but deliberate changes in the method parameters. The robustness of the method was determined
by deliberately varying pH, flow rate, and column temperature. The effect of mobile phase pH was
studied at 2.3 and 2.7, the effect of flow rate was studied at 0.29mL/min and 0.35mL/min, and the
effect of column oven temperature was studied at 30 and 40C. The impact of column temperature
on the assay of PLX was studied at 50 and 60C. System suitability parameters, such as USP resolu-
tion, tailing % RSD, and retention time, of PLX were noted. The data are listed in Table 7. The data
reveal that the method is sensitive to low pH and influences the resolution between impurity 1 and
PLX. Furthermore, the flow rate affects the retention behavior of PLX.

Table 6. Results of stability of standard solution for impurities at room temperature

Name % of impurities (w/w) for sample-1 % of impurities (w/w) for sample-2
of the Initial After 24h % Initial After 24h %
impurity Difference Difference
Impurity 1 0.061 0.058 4.9 0.070 0.068 2.9
Impurity 2 0.105 0.102 2.9 0.112 0.109 2.7
Impurity 3 0.051 0.050 2.0 0.050 0.050 0
Impurity 4 0.060 0.060 0 0.060 0.060 0
Difference Difference
Total 0.358 0.312 0.046 0.301 0.298 0.003
impurities

Table 7. Robustness and method sensitivity data

S. no. Condition RT of Assay of USP USP % RSD of Method
plerixafor plerixafor resolution tailing of standard sensitivity*
(min) in spiked for plerixafor
sample impurity 1
(%w/w)
1 Control (No 1.161 99.1 2.2 1.3 0.3 NA
change)
2 pH ()2.3 1.125 98.9 1.6 1.5 0.65 Yes
3 pH (+)2.7 1.105 98.9 2.2 1.3 0.44 No
4 Flow 1.086 99.1 1.9 1.2 0.51 Yes
(+)0.35mL/min
5 Flow 1.341 99.3 2.3 1.4 0.52 No
()0.29mL/min
6 Column oven 1.122 99.0 2.2 1.2 0.44 No
temperature
(+)40C
7 Column oven 1.163 99.0 2.0 1.2 0.48 No
temperature ()
(30C)
*Based on the comparison of results from altered parameters with those of control from.

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5. Conclusions
The rapid gradient reverse phase UPLC method, developed for the quantitative analysis of PLX and
related substances in pharmaceutical dosage forms, is precise, accurate, linear, robust, and specific.
Satisfactory results were obtained from validation of the method. The retention time (1.1min) ena-
bles rapid estimation of the drug. This method exhibits an excellent performance in terms of sensi-
tivity and speed. The method is stability-indicating and can be used for routine analysis of production
samples, checking the stability of samples, and checking the stability of samples of PLX.

Acknowledgments CSF vs. placebo+G-CSF in non-Hodgkins lymphoma

The authors wish to thank the management of Mylan patients forautologous hematopoietic stem cell (aHSC)
Laboratories Ltd for supporting this work. We would also like transplantation (Abstract No. 601). Blood, 110, 185a.
to thank colleagues in the analytical development services DiPersio J., Stadtmauer E. A., Nademanee A. P., Stiff, P., Micallef,
for their co-operation in carrying out this work. I., Angell, J., Calandra, G. (2007, November 16). A phase
III, multicenter, randomized, double-blind,
Funding placebocontrolled, comparative trial of AMD3100
This work was supported by the Mylan Laboratories Ltd. (plerixafor)+GCSF vs. G-CSF+placebo for mobilization in
multiple myeloma (MM) patients for autologous
Author details hematopoietic stem cell (aHSC) transplantation (Abstract
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