Sei sulla pagina 1di 6

Journal of Pharmaceutical and Biomedical Analysis 104 (2015) 4954

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

journal homepage:

Efcient HPLC method development using structure-based database

search, physico-chemical prediction and chromatographic simulation
Lin Wang a , Jinjian Zheng b, , Xiaoyi Gong a , Robert Hartman b , Vincent Antonucci a

Merck Research Laboratories, Merck, Rahway, NJ 07065, USA

Merck Manufacturing Division, Merck, Rahway, NJ 07065, USA

a r t i c l e

i n f o

Article history:
Received 11 July 2014
Received in revised form 17 October 2014
Accepted 31 October 2014
Available online 8 November 2014
Chromatographic simulation
Physico-chemical prediction

a b s t r a c t
Development of a robust HPLC method for pharmaceutical analysis can be very challenging and timeconsuming. In our laboratory, we have developed a new workow leveraging ACD/Labs software tools
to improve the performance of HPLC method development. First, we established ACD-based analytical
method databases that can be searched by chemical structure similarity. By taking advantage of the
existing knowledge of HPLC methods archived in the databases, one can nd a good starting point for
HPLC method development, or even reuse an existing method as is for a new project. Second, we used
the software to predict compound physicochemical properties before running actual experiments to
help select appropriate method conditions for targeted screening experiments. Finally, after selecting
stationary and mobile phases, we used modeling software to simulate chromatographic separations for
optimized temperature and gradient program. The optimized new method was then uploaded to internal
databases as knowledge available to assist future method development efforts. Routine implementation
of such standardized workows has the potential to reduce the number of experiments required for
method development and facilitate systematic and efcient development of faster, greener and more
robust methods leading to greater productivity. In this article, we used Loratadine method development
as an example to demonstrate efcient method development using this new workow.
2014 Elsevier B.V. All rights reserved.

1. Introduction
Analytical testing and control strategy plays a critical role
during the entire life cycle of the drug development process
in the pharmaceutical industry. HPLC is the major work horse
that has been used for all aspects of pharmaceutical analysis
including assay, dissolution analysis, impurity prole, forced degradation studies, process control, and drug metabolism studies
[14]. Given the time constraints and limited resources in an
R&D laboratory, it is imperative to develop robust HPLC methods
quickly to support the drug development process. Many different
approaches to improve the performance of HPLC method development have been reported [58]. Often, a column screening
system is used to nd a promising combination of mobile
phase/stationary phase which meets desired criteria, and the separation is subsequently optimized using a software tool such
as DryLab [712] or Chromsword [5,6,1316]. These software
tools allow the scientist to model chromatographic separations
based upon retention data from a limited number of scouting

Corresponding author. Tel.: +1 732 594 4515; fax: +1 732 594 3887.
E-mail address: (J. Zheng).
0731-7085/ 2014 Elsevier B.V. All rights reserved.

experiments, and optimal separation conditions can be predicted

by the modeling software. This approach avoids labor intensive
trial-and-error experiments, potentially resulting in signicant
improvement in method development efciency and nal method
In spite of the successes with software simulation, there are certain limitations. Typically, software simulation is done with one
stationary phase and one set of mobile phases. Therefore, it is critical to select the appropriate combination of stationary and mobile
phases for evaluation in scouting experiments. Screening experiments can help the scientist make informed decisions on good
stationary phase and mobile phase candidates. However, without a
good understanding of the physicochemical properties of analytes
such as pKa , log P, log D, and solubility, these screening experiments may not yield desired results within a reasonable time frame
due to excessive trial-and-error experimentation. Ultimately, the
chromatographic experience of the scientist is as important a
factor in overall success as the automation and simulation tools
mentioned above. Therefore, we think that the optimal workow for efcient method development must thoughtfully combine
elements of knowledge management, software physicochemical
property prediction, chromatographic simulation, and focused


L. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 104 (2015) 4954

2.3. Materials and reagents

Acetonitrile (HPLC grade), water (HPLC grade), sodium hydroxide solution (10 N) and triethylamine (HPLC grade) were purchased
from Fisher Scientic (Pittsburgh, PA, USA). Phosphoric acid (85 wt%
in H2 O, 99.99% trace metal basis) and boric acid (99.99%) were purchased from SigmaAldrich (St. Louis, MO, USA). Loratadine and
impurities were synthesized by Merck Sharp & Dohme Corp. (Rahway, NJ, USA).
2.4. Chromatographic conditions
Detailed chromatographic conditions are provided in specic
gure captions.
Fig. 1. A new strategy for efcient HPLC method development.

3. Results and discussion

Herein, we propose an integrated workow for HPLC method
development shown in Fig. 1. First, we integrated existing
screening results, method information, and vendor applications
into structure-searchable databases. Knowledge and information
is preserved and re-used to expedite method development. By
searching structure similarity, one can quickly identify a good starting point (such as column, pH, and mobile phase) for target analytes.
Following that, we further leveraged software tools to predict compound physico-chemical properties such as pKa /log P/log D before
running actual experiments. Such information serves as a rule of
thumb to help select appropriate chromatographic techniques as
well as method conditions such as stationary phase, buffer pH,
and mobile phase additives for targeted and focused screening.
After the screening experiments to dene the stationary and mobile
phases, a few scouting runs are performed to experimentally verify the in silico selectivity predictions, and separation modeling
software (e.g. ACD/Labs, LC simulator, DryLab, etc.) was used to
simulate chromatographic separations for rapid development of
faster, greener, and more robust methods. With the completion of
method optimization, the new method is introduced to the internal database to assist future method development for a structurally
similar compound. In this article, we will use an example of Loratadine method development to illustrate how we have successfully
combined a structure-searchable database tool, physico-chemical
prediction tool, and LC simulator to create a holistic workow for
efcient HPLC method development and lifecycle management.
Further, the workow presented also supports a shift from extensive high-throughput laboratory screening as the basis of method
development, to a model where knowledge retrieval, prediction,
and simulation suggest optimal analysis conditions which only
require limited experimental verication in the laboratory before
2. Experimental
2.1. HPLC instrumentation
All experiments were conducted on Agilent HPLC 1100 systems
(Agilent, Santa Clara, CA, USA) equipped with an autosampler, a
quaternary pump and a variable wavelength detector. The maximum operating pressure of the system was 400 bar. Empower II
software (Waters, Milford, MA, USA) was used to control the HPLC
system and for data acquisition and analysis.
2.2. HPLC columns
XBridge C18 columns (100 mm 4.6 mm I.D., 3.5 m) were purchased from Waters, Milford, MA, USA.

3.1. Search databases for starting point of method development

To improve productivity and reduce R&D costs in an analytical laboratory, signicant emphasis is placed on high-throughput
and multiplexed analyses aimed at generating many experimental results in a short period of time. However, one aspect of the
method development process that is often overlooked is leveraging past experiments to inform current experiments, or simply
using the power of knowledge management and prediction to assist
high-throughput experimentation. As an example, methods are
often developed for commonly observed analytes (e.g. solvents and
reagents) simultaneously within individual groups of a large scientic organization, resulting in signicant duplication of effort,
and the best methods available are not always being implemented
in each laboratory. Development of optimal analytical methods
often requires a high level of technical expertise, particularly for
challenging low level quantitative analyses such as those used for
mutagenic impurities [17]. For many organizations, a major inefciency is the one-and-done data life cycle where insights are only
captured in the heads of individual scientists and not shared, or
these insights are stored in an electronic repository with limited
access across the organization.
One way to address this problem is to provide a platform for
research scientists to capture and share their knowledge by combining live analytical data with chemical and structural context. In
the present research, we have leveraged the commercially available
ACD/Web Librarian software to establish web-accessible databases
that are searchable by structure. Our current analytical method
databases include achiral and chiral separation methods obtained
from application notes provided by column manufacturers, literature resources, and analytical methods generated within the
company. All databases are updated regularly. One major advantage of establishing structure searchable databases is that users
are able to search by chemical structure similarity, which is not
feasible by text-based searches. Analytical methods for a structurally similar compound can provide a promising starting point
for method development when a method for the exact compound
is not available.
As a model system to evaluate the integrated workow in our
laboratory, development of an impurity/degradate HPLC prole for
Loratadine was selected. Shown in Fig. 2 are the structures of potential Loratadine related impurities or degradation products. These
compounds are either listed in compendial monograph (e.g. USP,
EP), or identied in the synthetic process. Separation of Loratadine
and its related compounds has been reported [18]. However, several key USP and EP specied impurities were not included in the
existing publications. Therefore, a new method that is capable of
separating all impurities listed in Fig. 2 is desired.

L. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 104 (2015) 4954


Fig. 2. Structures of Loratadine (compound J) and its related compounds. Compound P is pseudoephedrine that is commonly used in combination with Loratadine. Compound
F is a pseudoephedrine related compound.

Initiating the proposed workow, databases were searched for

structure similarity (both Markush and simple structure). There are
no records for the separation of exact compounds in the database.
However, we found multiple records for the separation of compounds with similar structures such as protriptyline, nortriptyline,
amitriptyline, doxepin, imipramine, desipramine, trimipramine,

and nordoxepin as shown in Fig. 3. The results from several key

applications were summarized in Table 1. Most of these methods
use either C18 or Cyano stationary phase. We chose C18 over Cyano
for method development due to its superior column stability, which
is critical for a commercial supply lab. Buffers with a wide pH range
from 2.7 to 12.0 have been used as shown in Table 1. Therefore, a

Fig. 3. Structures of compounds with similar structures to Loratadine found in the databases including protriptyline, nortriptyline, amitriptyline, doxepin, imipramine,
desipramine, trimipramine, and nordoxepin.


L. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 104 (2015) 4954

Table 1
Summary of methods for similar structures from application database.


Mobile phase


Protriptyline, nortriptyline,
doxepin, imipramine,

Purospher STAR RP-18

Water/MeOH/potassium phosphate, pH 7.6

Good resolution. Peak tailing

Protriptyline, nortriptyline,
doxepine, imipramine,
amitriptyline, trimipramine


Water/acetonitrile/TEA-phosphate, pH 6.9

Peak tailing of early eluting


Doxepine, trimipramine,
amitriptyline, imipramine,
nordoxepin, nortriptyline,
desipramine, protriptyline

Ascentis ES Cyano

Acetonitrile, methanol, potassium phosphate, pH 7

Good peak shape and


Doxepine, protriptyline,
imipramine, nortriptyline,
amitriptyline, trimipramine

Hypersil Gold

Acetonitrile/water/0.1% formic acid, pH 2.7

Weak retention. Peak tailing

Nordoxepin, protriptyline,
nortriptyline, imipramine,


Acetonitrile/water/potassium phosphate, pH 12.0

Good peak shape and


Desmethyldoxepin, protriptyline,
esipramine, nortriptyline,
doxepine, imipramine,
amitriptyline, timipramine

Pursuit C18

Water/methanol/acetonitrile/potassium phosphate,
pH 7.0

Good peak shape

Doxepine, imipramine,
nortriptyline, amitriptyline,

Zorbax Extend C18

Water/MeOH pyrrolidine buffer, pH 11.5

Good separation and peak


Nortriptyline, imipramine,
amitriptyline, clomipramine

XBridge C18

Acetonitrile/methanol/sodium phosphate, pH 7.0

Excellent peak shape and


column that is stable across a wide pH range is preferred. A Waters

Xbridge C18 column was selected because it shows excellent peak
shape and separation for similar compounds, and it is stable across
a wide pH range due to its unique ethylene bridged hybrid silica. In
several database applications, 0.1% formic acid was used as mobile
phase modier. We replaced it with 0.1% phosphoric acid to reduce
baseline noise. Acetonitrile was selected because it was used in the
compendial method and a relevant literature [18] for Loratadine
and its related compounds.
From preliminary injections using the selected stationary and
mobile phases, severe tailing of Loratadine peak was observed as
shown in Fig. 4. The tailing factors range from 1.11 for 0.005 mg/mL
sample solution to 3.54 for 0.5 mg/mL sample solution. One possible root cause is the overloading of protonated Loratadine because
ionized analyte usually has much less loading capacity [19]. As
a result, the retention time shifted from 5.56 min for 0.5 mg/mL

Fig. 4. Overlaid chromatograms of Loratadine API at different concentrations: (a)

0.5 mg/mL, (b) 0.05 mg/mL, and (c) 0.005 mg/mL. HPLC column: Waters XBridge
C18, 3.5 m, 100 mm 4.6 mm; injection volume: 5 L. Mobile phases: (A) 0.1%
H3 PO4 and (B) acetonitrile. Flow rate: 1.5 mL/min; temperature: 35 C; detection:
UV absorbance at 270 nm; gradient: 010 min, 2550%B.

solution to 5.74 min for 0.05 mg/mL solution, and 5.81 min for
0.005 mg/mL solution (the lower the concentration, the longer
the retention time). Therefore, it is difcult to identify peaks
by RRT (relative retention time) as the retention time of active
pharmaceutical ingredient (API) varies with concentration. Clearly,
choice of appropriate buffer pH is critical to achieve a good peak
shape, highlighting the value of rst understanding basic analyte
physico-chemical properties prior to conducting generalized, broad
parameter screens to avoid collection of extensive data of limited

3.2. Select suitable buffer pH

For the separation of ionic analytes, controlling mobile phase
pH is critical to the performance of the method. Typically, the pH
should be chosen at least 1.5 unit away from pKa s of all components if possible [20]. Although pKa could be estimated by checking
the functional groups in molecules, this approach is not very accurate due to electronic and/or steric effects. Using pKa calculation
software, however, pKa values can be calculated with an accuracy
of 0.3 or better in most cases.
Employing the proposed integrated workow, the pKa of Loratadine is calculated to be 4.27 by ACD software. Based on the rule
stated above, a basic buffer (20 mM boric acid with 0.02% triethylamine (TEA), pH adjusted to 9.5 with sodium hydroxide) was used
to replace the 0.1% phosphoric acid as mobile phase A. As shown
in Fig. 5, excellent peak shape (tailing factor ranging from 1.03 to
1.05) and constant retention time (retention time ranging from
8.19 min to 8.21 min) were observed for Loratadine even though
its concentration varies from 0.005 mg/mL to 0.5 mg/mL, thus signicantly improving the consistency of peak identication. With
this physico-chemical property prediction in hand, we are now better positioned to conduct focused experimental verication runs
using the selected chromatographic mobile phase and column to
optimize the method more efciently.

L. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 104 (2015) 4954

Fig. 5. Overlaid chromatograms of Loratadine API at different concentrations: (a)

0.5 mg/mL, (b) 0.05 mg/mL, and (c) 0.005 mg/mL. HPLC column: Waters XBridge C18,
3.5 m, 100 mm 4.6 mm; injection volume: 5 L. Mobile phases: (A) 20 mM boric
acid + 0.02% triethylamine (TEA), pH adjusted to 9.5 with sodium hydroxide and (B)
acetonitrile. Flow rate: 1.5 mL/min; temperature: 35 C; detection: UV absorbance
at 270 nm; gradient: 010 min, 3565%B.

3.3. Optimize separation

After selection of stationary phase, pH, and organic modier, the
next step is to optimize gradient and temperature. Traditional trialand-error experimentation is usually time-consuming, and may
not be able to yield the optimal method, especially for complex
samples as the one used in this study. Computer software simulation can help us evaluate separation under new conditions within
minutes, which improves the efciency and makes it possible to
develop a robust method within a reasonable time frame. Computer
software assisted HPLC method development has been reported
since the late 1970s [1,21]. Currently, a number of software programs are commercially available such as DryLab (LC Resources,
USA), ChromSword (Merck KGaA, Germany), Fusion AE (S-Matrix,
USA), and ACD/Labs (Advanced Chemistry Development, Canada),
and there are many reports on fast method development using
such software [2226]. Typically, scouting runs (e.g. 2 runs for
gradient or 4 runs for gradient/temperature modeling) are rst
acquired in the laboratory and used to build a retention model.
Resolution maps show the effect of a given variable on the separation of a set of compounds and clearly describe the resolution
of the critical pair (least-resolved components) as a function of
a given variable. The computational results are then utilized as
guidance for further experimental work in the laboratory. This procedure is repeated until satisfactory chromatography is achieved.
The chromatogram of the optimized method was shown in Fig. 6.


Fig. 7. Chromatogram for assay and content uniformity of Loratadine. Column:

Waters XBridge C18, 3.5 m, 100 mm 4.6 mm. Mobile phases: (A) 20 mM boric
acid + 0.02% triethylamine (TEA), pH adjusted to 9.5 with sodium hydroxide and (B)
acetonitrile. Temperature: 35 C; detection: UV absorbance at 270 nm; ow rate:
1.5 mL/min; injection volume: 5 L. Isocratic at 59%B. Run time: 5 min.

All Loratadine related compounds were baseline separated from

each other and from Loratadine with a minimum resolution of 1.5.
Therefore, this method can be used as an impurity proling method
for Loratadine drug substance, or as a stability indicating method
for Loratadine drug product.
3.4. Manage and reuse knowledge
The method shown in Fig. 6 was uploaded to the database,
and was later applied to several different formulations including
tablets, syrup and for different purposes such as stability testing,
dissolution, and content uniformity with minor modications. For
the content uniformity and dissolution testing, the goal is to separate the API from other impurities, but it is not necessary to separate
impurities from each other. The challenge, however, lies in the large
sample size. For example, a dissolution test is typically conducted in
multiple vessels (e.g. n = 624) with samples taken at multiple time
points for each batch [27]. To determine the content uniformity of
the dosage form, about 1030 samples are analyzed per batch [28].
Therefore, fast separation is desired. Using the retention model
built for the impurity proling method shown in Fig. 6, we were
able to quickly simulate a fast isocratic method to separate Loratadine from all other impurities. The experimental chromatogram
was shown in Fig. 7. Loratadine was baseline separated from all
other related compounds within 5 min with a minimum resolution
of 2.0, which signicantly improved the throughput.
4. Conclusions

Fig. 6. Chromatogram of Loratadine and its related compounds. Column: Waters

XBridge C18, 3.5 m, 100 mm 4.6 mm. Mobile phases: (A) 20 mM boric acid + 0.02%
triethylamine (TEA), pH adjusted to 9.5 with sodium hydroxide and (B) acetonitrile.
Temperature: 35 C; detection: UV absorbance at 270 nm; ow rate: 1.5 mL/min;
injection volume: 5 L. Gradient: 015 min, 2535%B; 1535 min, 3565%B.

We have presented an effective workow for method development employing a combination of hardware, software, focused
experimentation, and application of personal and organizational
knowledge. The new workow consists of three parts. First,
we established ACD-based HPLC method databases that can be
searched by chemical structure similarity to take advantage of the
existing knowledge of HPLC methods archived in the databases.
Second, we used software to predict compound physico-chemical
properties before running actual experiments to help select appropriate method conditions for targeted screening experiments. It is
worth noting that for Loratadine method development, screening
of stationary phase and mobile phases were not performed as all
impurities were well characterized, and a good starting condition
was found from the database. For complex samples where there
are lots of unknown impurities, targeted screening may be necessary to nd a promising starting condition. Finally, we use the


L. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 104 (2015) 4954

modeling software to simulate chromatographic separations, followed by targeted experimental verication in the laboratory, to
rapidly develop fast and robust HPLC methods. The optimized new
method was then added to the databases to assist future method
development. Such workow brings the benet of reduction in the
number of experiments required for method development. Faster,
greener and more robust methods are developed in a systematic and efcient manner, leading to productivity gains in overall
method development.
The authors would like to thank Thom Loughlin, Yong Liu,
Xiaodong Bu, and Patrick Chin for their support on Merck internal structure-searchable databases; and thank Mary Rogowski and
Karim Kassam from ACD/Labs for their support.
[1] L.R. Snyder, J.J. Kirkland, J.L. Glajch, Practical HPLC Method Development, John
Wiley & Sons, Inc., 1997.
[2] G. Lunn, N.R. Schmuff, HPLC Methods for Pharmaceutical Analysis, vols. 14,
John Wiley, New York, 19972000.
[3] G. Lunn, HPLC Methods for Recently Approved Pharmaceuticals, Wiley Interscience, New York, NY, 2005.
[4] J. Swadesh, HPLC Practical and Industrial Applications, CRC Press Inc., Boca
Raton, FL, USA, 1997.
[5] K.P. Xiao, Y. Xiong, F.Z. Liu, A.M. Rustum, Efcient method development strategy for challenging separation of pharmaceutical molecules using
advanced chromatographic technologies, J. Chromatogr. A 1163 (2007)
[6] J. Zheng, A.M. Rustum, Rapid separation of desloratadine and related
compounds in solid pharmaceutical formulation using gradient ion-pair chromatography, J. Pharm. Biomed. Anal. 51 (2010) 146152.
[7] R.M. Krisko, K. McLaughlin, M.J. Koenigbauer, C.E. Lunte, Application of a
column selection system and DryLab software for high-performance liquid chromatography method development, J. Chromatogr. A 1122 (2006)
[8] K. Jayaraman, A.J. Alexander, Y. Hu, F.P. Tomasella, A stepwise strategy employing automated screening and DryLab modeling for the development of robust
methods for challenging high performance liquid chromatography separations:
a case study, Anal. Chim. Acta 696 (2011) 116124.
[9] I. Molnar, Computerized design of separation strategies by reversed-phase liquid chromatography: development of DryLab software, J. Chromatogr. A 965
(2002) 175194.

[10] L.R. Snyder, J.W. Dolan, D.C. Lommen, Drylab computer simulation for highperformance liquid chromatographic method development. I. Isocratic elution,
J. Chromatogr. 485 (1989) 6589.
[11] J.W. Dolan, D.C. Lommen, L.R. Snyder, Drylab computer simulation for
high-performance liquid chromatographic method development. II. Gradient
elution, J. Chromatogr. 485 (1989) 91112.
[12] I. Molnr, K. Monks, From Csaba Horvth to quality by design: visualizing
design space in selectivity exploration of HPLC separations, Chromatographia
73 (Suppl. 1) (2011) S5S14.
[13] S.V. Galushko, A.A. Kamenchuk, G.L. Pit, Calculation of retention in reversedphase liquid chromatography. IV. ChromDream software for the selection
of initial conditions and for simulating chromatographic behaviour, J. Chromatogr. A 660 (1994) 4759.
[14] W.D. Beinert, R. Jack, V. Eckert, S. Galushko, V. Tanchuk, I. Shishkina, A program
for automated HPLC method development, Am. Lab. 33 (2001) 1415.
[15] E.F. Hewitt, P. Lukulay, S. Galushko, Implementation of a rapid and automated
high performance liquid chromatography method development strategy for
pharmaceutical drug candidates, J. Chromatogr. A 1107 (2006) 7987.
[16] S.V. Galushko, A.A. Kamenchuk, G.L. Pit, Software for method development in
reversed-phase liquid chromatography, Am. Lab. 27 (1995) 421432.
[17] Andrew Teasdale, Genotoxic Impurities Strategies for Identication and Control, John Wiley & Sons, Inc., 2010.
[18] J. Lu, Y.C. Wei, R.J. Markovich, A.M. Rustum, The retention behavior of Loratadine
and its related compounds in ion pair reversed phase HPLC, J. Liq. Chromatogr.
Related Technol. 33 (2010) 603614.
[19] J. Dai, W.C. Peter, McCalleyF D.V., A new approach to the determination of
column overload characteristics in reversed-phase liquid chromatography, J.
Chromatogr. A 1216 (2009) 24742482.
[20] A.N. Heyrman, R.A. Henry, Importance of Controlling Mobile Phase pH in
Reversed Phase HPLC, Keystone Technical Bulletin, TB 99-06, 1999.
[21] T. Baczek, R. Kaliszan, H.A. Claessens, M.A. van Straten, Computer-assisted optimization of reversed-phase HPLC isocratic separations of neutral compounds,
LCGC Europe (June) (2001) 26.
[22] J.L. Glajch, L.R. Snyder (Eds.), Computer-assisted Development for High Performance Liquid Chromatography, Elsevier, Amsterdam, 1990 (J. Chromatogr. 485
[23] R.S. Hodges, J.M. Parker, C.T. Mant, R.R. Sharma, Computer simulation of
high-performance liquid chromatographic separations of peptide and protein
digests for development of size-exclusion, ion-exchange and reversed-phase
chromatographic methods, J. Chromatogr. 458 (1988) 147167.
[24] R.C. Chloupek, W.S. Hancock, L.R. Snyder, Computer simulation as a tool for the
rapid optimization of the high-performance liquid chromatographic separation
of a tryptic digest human growth hormone, J. Chromatogr. 594 (1992) 6573.
[25] T.H. Hoang, D. Cuerrier, S. McClintock, M. Di Maso, Computer-assisted method
development and optimization in high-performance liquid chromatography, J.
Chromatogr. A 991 (2003) 281.
[26] M. Meng, L. Rohde, V. Capka, S.J. Carter, P.K. Bennett, Fast chiral chromatographic method development and validation for the quantitation of eszopiclone
in human plasma using LC/MS/MS, J. Pharm. Biomed. Anal. 53 (2010) 973982.
[27] US Pharmacopeia, Chapter 711, Dissolution.
[28] US Pharmacopeia, Chapter 905, Uniformity of Dosage Units.