CHIRALITY 17:S24–S29 (2005)

Analysis of Chiral Pharmaceuticals Using HPLC

With CD Detection
AMANDA L. JENKINS* and WILLIAM A. HEDGEPETH
Jasco Inc., Easton, Maryland

ABSTRACT Recent American Food and Drug Administration guidelines have effec-
tively determined that mixtures of chiral compounds can no longer be brought to the
pharmaceuticals marketplace. These guidelines require a means for chiral detection
and compound separation. This article describes separation and detection of chiral
pharmaceuticals using HPLC with circular dichroism detection. Two over-the-counter
(OTC) medications, Prilosec and naproxen sodium, along with the prescription drugs
Naproxen, Nexium, and Coumadin were analyzed using HPLC with CD, UV, and optical
rotation (OR) detection. In all cases the CD and UV detectors displayed a much greater
sensitivity than the OR detector. Although in many cases the UV detector did have a
slightly better sensitivity than the CD detector, the CD detector had the advantage of
only responding to the chiral compounds, eliminating the possibility of interference
with the peaks of interest. Chirality 17:S24 – S29, 2005. A 2005 Wiley-Liss, Inc.

KEY WORDS: chirality; separation; chromatography; drugs; detection

In the early 20th century the relevance of chirality to the
pharmaceutical industry was established by the fact that
one enantiomer of hyoscyamine possessed greater phar-
macological activity than the other. Today, most new
drugs and those under development consist of a single
optically active isomer, and chirality is also becoming an
issue for the agrochemical and other industries. Regu-
latory agencies throughout the world are currently
reviewing the importance of chirality with regard to phar-
maceutical and agrochemical products. New guidelines
from such agencies have been key drivers for the focus on
single enantiomer products in these industries.
There are many chiral issues in drug development.
Racemic drugs can cause problems because of the dif-
ferences not only in the biological effects but also in the
pharmacokinetics of the enantiomers. One example is
Perhexiline, a drug used to treat abnormal heart rhythms.
In the 1980s the racemate killed a number of people who
had accumulated gram quantities of the enantiomer that
was more slowly metabolized. If researchers had realized
that one enantiomer had a much longer half-life, they
might have come up with a drug with just the fast-clearing
enantiomer. It was one of the very early examples where
an understanding of chirality in drugs is critical.1
At present, a number of chiral switches are still in the
pipeline. What is likely to increase is the development
of single enantiomers of a chiral compound for different
therapeutic uses, as exemplified by the dextromethorpan/
levomethorpan dichotomy. Examples include the work on
methylphenidate (Ritalin), a drug to treat attention defi-
cit hyperactivity disorder (ADHD). Methylphenidate has
two chiral centers and the drug is a racemate of the S,S
and R,R isomers. Natural products present another fertile
A 2005 Wiley-Liss, Inc.
field for discovering therapeutic properties of single en-
antiomers. Recent studies of the enantiomers of natural
products or semisynthetic drugs derived from natural
products indicate that they could have distinct thera-
peutic value. For example, ( – )-quinine is an antimalarial,
whereas its quasienantiomer (+)-quinidine is an antiar-
rhythmic compound.2
Most of the pharmaceutical and pharmacological
studies of stereoselectivity of chiral drugs before the
mid-1980s involved precolumn derivatization of the enan-
tiomers with chiral reagents, forming diastereomers. The
diastereomers were subsequently separated in the nor-
mal or reversed phase mode of chromatography.3 Great
efforts have been devoted to the development of better
methodology for enantioselective chromatography during
the past decade and have resulted in new chiral stationary
phases, pioneered by Pirkle.4 Chiral agents were deriva-
tized and immobilized on the surface of the support (silica
gel mostly), and served as the in situ chiral discriminators
during the chromatographic process. The preference of
chiral stationary phases lies in the inherent advantages
of any chromatographic separation, such as the speed
of the analysis, the possibility to analyze or purify the
enantiomers in complex mixtures, and the reproducibility
of the analysis and its flexibility. Moreover, analytical
chromatographic systems can be adapted to preparative
*Correspondence to: A.L. Jenkins, Jasco Inc., 8649 Commerce Dr., Easton,
MD 21601. E-mail: Jenkins@jascoinc.com
Received for publication 14 July 2004; Accepted 25 October 2004
DOI: 10.1002/chir.20104
Published online in Wiley InterScience (www.interscience.wiley.com).
 ANALYSIS OF CHIRAL PHARMACEUTICALS
separations, in which pure enantiomers can be collected.5,6
In addition to their distinct practical applicability, chiral
stationary phases can uniquely contribute to studies of
the nature of molecular recognition. Since the differential
retention of enantiomers in the chromatographic system
employing chiral stationary phases can be attributed only
to chiral discrimination by the chiral sites, these inter-
actions can be isolated and explored. It has been shown
that chromatographic parameters obtained by chiral sta-
tionary phases can be sensitive to very subtle differences
between the enantiomers. Moreover, chiral stationary
phases can be tailor-made to accommodate specific stud-
ies of chiral recognition between molecules.
Several detectors are available that can selectively de-
tect chiral compounds. Two of these are the optical ro-
tation (OR) and the circular dichroism detector (CD).
Optical rotatory dispersion (OR) detection is based on the
refractive index difference between right and left linearly
polarized lights. Unlike many detectors for HPLC, the OR
is a universal detector (does not require a chromophore).
It is most commonly used for analysis of sugars. Since it
is based on refractive index changes, the measurement is
subject to temperature and solvent changes and thus has
difficulties in doing gradients. CD detection is based on
an absorption difference between right and left circularly
polarized light. This type of detection is intrinsically stable
Fig. 1. Naproxen chromatograms. Top, CD detector; middle, UV detector; bottom, OR detector.
S25
during temperature and solvent changes, making it gra-
dient compatible. Since this is an absorption-based tech-
nique, the material being studied must have a chromophore
for detection. This article briefly describes the detection
of several chiral pharmaceuticals and compares and con-
trasts the different detectors used in HPLC.
MATERIALS AND METHODS
A prescription Naproxen tablet (492.7 mg) and an over-
the-counter naproxen sodium tablet (288.2 mg) were sep-
arately ground. Ten mL ethanol was added to the
naproxen sodium tablet and 20 mL ethanol was added to
the naproxen tablet. Both samples were sonicated for
15 min. These solutions were then diluted to a final con-
centration of 5 mg/mL and filtered through a 0.45 mm
PVDF syringe filter. A Jasco HPLC system equipped with
a PU-2089 quaternary gradient pump, an AS-1559 auto-
sampler, a CD-1595 circular dichroism detector, and an
OR-1590 optical rotation detector was used. Injection vol-
umes for both samples were 1 mL. Separations were per-
formed with a ChiralPak AD column (4.6  250 mm)
(Chiral Technologies, Eaton, PA) using a mobile phase
of 100% ethanol (EtOH) and a modifier of 0.1% TFA (tri-
fluoroacetic acid) with a flow rate of 1.0 mL/min. CD/UV
data were monitored at 260 nm.
S26 JENKINS AND HEDGEPETH

Fig. 2. Naproxen-sodium chromatograms. Top, CD detector; middle, UV detector; bottom, OR detector.

Fig. 3. Nexium chromatograms. Top, CD detector; middle, UV detector; bottom, OR detector.

 ANALYSIS OF CHIRAL PHARMACEUTICALS
Prescription Nexium and Prilosec were also examined
using the HPLC system. A 409 mg Prilosec tablet con-
taining 20 mg omeprazole was ground, 20 mL ethanol
added, and the resulting solution sonicated for 20 min.
Similarly, a 462 mg Nexium tablet reported to contain 20 mg
esomeprazole was ground, 20 mL of ethanol added, and
sonicated for 30 min. The resulting solutions were brought
to a final concentration of 1 mg/mL after filtration and 2 mL
injections were made. The solutions were analyzed using
a ChiralPak AD column (Chiral Technologies; 4.6 
250 mm) with a 100% EtOH mobile phase and a flow rate
of 0.9 mL/min. CD/UV data was monitored at 290 nm.
Warfarin sodium, more commonly known as Coumadin,
was also evaluated as it can be used as both a phar-
maceutical and a pesticide as a result of its blood-thinning
ability. A 202.1 mg prescription tablet reported to contain
5 mg of the drug was ground. Five mL of ethanol was
added and sonicated for 20 min. The solution was filtered
and 1-mL injections were made. The separation was per-
formed with a ChiralPak AD column (4.6  250 mm; Chiral
Technologies). A mobile phase of 100% ethanol (EtOH)
and a modifier of 0.1% TFA (tri-fluoroacetic acid) was run
with a flow rate of 1.0 mL/min. CD/UV data were mon-
itored at 310 nm.
Fig. 4. Prilosec chromatograms. Top, CD detector; middle, UV detector; bottom, OR detector.
S27
RESULTS AND DISCUSSION
Naproxen is a member of the arylacetic acid group of
nonsteroidal antiinflammatory drugs. It is lipid soluble,
practically insoluble in water at low pH, and freely soluble
in water at high pH.7 Naproxen is used to relieve the pain,
tenderness, inflammation (swelling), and stiffness caused
by gout, arthritis, and other inflammatory conditions. It
also is used to relieve other pain, including muscle and
menstrual pain and pain after surgery, dental work, or
childbirth. Enantiomeric purity in naproxen is very im-
portant, as only the (S) isomer is safe. The (R) isomer is
reported to be a liver toxin. Researchers have also shown
that the (S) chiral form of the drug naproxen has 28 times
the antiinflammatory activity of the opposite chiral rela-
tive. Another interesting feature of naproxen is that the
free acid is dextrorotatory; merely neutralizing it makes
the sodium salt (the material commercially distributed),
which is levorotatory. This offers a striking example of
the lack of obvious relationship between structure and
optical rotation, since both acid and salt have the same
configuration about the chiral center.8
The chromatograms of Naproxen and the naproxen
sodium are shown in Figures 1 and 2. Both the Naproxen
S28 JENKINS AND HEDGEPETH
and the sodium salt showed only one peak in the CD
chromatogram, as expected. This indicates that both the
prescription and the over-the-counter versions are meet-
ing the requirements to be enantiomerically pure. As seen
in both figures the UV detector is the most sensitive,
about 10 times more sensitive than the CD detector and
about 100 times more sensitive than the OR detector.
Since these are relatively clean samples, UV or CD would
be the method of choice. However, if this was a complex
sample and only the chiral peaks were of interest, the
CD detector would be better since it only responds to the
chiral compounds.
Nexium was one of the first pharmaceuticals to be re-
patented and marketed as a pure enantiomer of a formerly
racemic drug. Nexium (esomeprazole) is the S-enantiomer
of omeprazole, the active ingredient in Prilosec, a drug
used to treat ulcers, heartburn, acid reflux, or Zollinger-
Ellison syndrome. Omeprazole was found to have signifi-
cant interindividual variability, while esomeprazole had a
superior clinical efficacy due to its higher and more con-
sistent bioavailability.9
The chromatograms for the Nexium and Prilosec are
shown in Figures 3 and 4. As expected, the CD of Prilosec
shows the inclusion of both enantiomers, while in Nexium
only the esomeprazole enantiomer is detected. The UV
detector was most sensitive for both materials, closely
followed by the CD detector. However, in the case of
Fig. 5. Warfarin sodium chromatograms. Top, CD detector; middle, UV detector; bottom, OR detector.
Prilosec the CD has a much cleaner baseline, as it
only responds to the chiral materials. Several small
peaks are seen in the baseline of the UV detector between
the 4 – 5-min retention time period. Although in this case
the peaks are small and do not interfere with detection,
it does show how in some cases the CD detector would
be crucial. The OR detector was also able to detect both
species, but its sensitivity is drastically lower than the
other detectors.
Anticoagulants, or blood thinners, such as Coumadin,
act to prevent blood from clotting. They will not break up
existing blood clots, nor will they literally ‘‘thin’’ your
blood. They should not be used by individuals with
bleeding problems like hemophillia or ulcers. Coumadin
is used to treat acute pulmonary embolism, atrial fibril-
lation (prevents clotting of the blood inside the heart),
deep vein thrombosis, prevents clotting after the replace-
ment of a heart valve, and is used after a heart attack or
stroke to prevent a recurrence. Coumadin has also been
used as a rodenticide since the 1940s. One advantage of
warfarin is that its effectiveness is based on proportion-to-
body-weight. Thus, a 1 kg rat or a 200 mg mouse gets a
fatal dosage that might not be fatal to, and perhaps not
even dangerous to, a 15 kg dog or 30 kg small child.10
One of the problems with warfarin is that the process
that manufactures it tends to leave a number of delete-
rious impurities in the resulting product. When used as a
 ANALYSIS OF CHIRAL PHARMACEUTICALS
rodenticide, this is not a major consideration, and these
are only dangerous over a sustained period of time. But it
was also determined that a good anticoagulant for human
beings would be a ‘‘Good Thing.’’ The product trade-
marked Coumadin is a form of crystalline sodium warfarin
that is created by a process that leaves no such impurities,
and therefore is safe for long-term human consumption.
Of course, since the patents on Coumadin have expired,
many other firms now synthesize the medication and call
it by its generic name, ‘‘sodium warfarin.’’ However, do not
think that if you are on Coumadin/warfarin therapy that
you can substitute rat poison; besides the impurities found
in the warfarin used for that purpose, many other po-
tentially fatal ingredients are used to enhance the effec-
tiveness of the rodenticide, and none of these would be
healthy to consume.11
The chromatogram from the prescription Coumadin tab-
let is shown in Figure 5. No peaks are observed with the
OR detector due to its decreased sensitivity in compar-
ison to the CD chiral detector.
CONCLUSIONS
HPLC with CD detection is an excellent method for the
separation and detection of chiral compounds, including
pharmaceuticals. Both the UV and the CD detectors were
S29
very sensitive with respect to concentration; however, the
UV detector cannot give any indication as to which enan-
tiomer is which. The optical rotation detector (ORD) is
capable of discriminating the enantiomers, but in all the
materials studied was found to be much less sensitive. In
general, the best compounds for use with the ORD are
those lacking a chromophore. Jasco currently offers the
world’s only CD detector for HPLC.
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