2.1 INTRODUCTION
The analysis of impurities in drug substances, beginning with the initial
screening and ending with the use of validated methods in routine quality control
and quality assurance, is becoming an increasingly challenging task along the
pharmaceutical value delivery chain. This compendium offers guidance on how
Rapid Resolution Liquid Chromatography (RRLC), in combination with mass
spectrometry, can improve overall workflow for analyzing and identifying
impurities in drug substances.
The pharmaceutical industry routinely uses high performance liquid
chromatography (HPLC) to quantitative levels of impurities at the mandated level
of 0.1% [l-31. However, pharmaceutical drug substances and their impurities are
often structurally similar and therefore may co-elute. The evaluation of peak
purity is a key component of method development and validation (i.e. specificity).
Analytical techniques typically used to increase the analyst's confidence in the
purity of chromatographic peaks include orthogonal separations, selectivity testing
using potential impurities and fraction collections followed by alternate testing.
These off-line techniques can be time consuming and ineffective, especially at the
early stages of drug development when impurities are unknown. Diode array
detection (DAD) has also been widely used to provide chromatographic peak
purity determinations [4-81. Although it was demonstrated that DAD can provide
rapid on-line determinations, disadvantages such as inability to consistently detect
an impurity below 0.5 per cent co-eluting with a high level main analyte and a
requirement for some separation of analytes were acknowledged. Analytes must
also have dissimilar UV spectra to achieve low detection levels. This shortcoming
is important in the pharmaceutical industry as impurities typically have UV
spectra similar to the main analyte. Coupling HPLC with mass spectrometry (LC-
MS) offers an alternative mode of detection which might be exploited for rapid
on-line HPLC peak purity assessment. Unlike DAD, LC-MS has the potential,
with the exception of isobaric impurities, to provide detection capabilities for all
impurities. Mass spectrometric detection offers the added ability to provide mass
and structural information on the co-eluting impurity. LC-MS can provide high
sensitivity for a wide range of compound classes relevant to the pharmaceutical
industry while simultaneously providing chromatographic selectivity and mass
selectivity. Indeed, LC-MS has been used to demonstrate method specificity and
to screen drug purity [9,10]. MS has also been implemented as a tool to determine
HPLC peak purity during the analysis of a few particular compounds [ll-141.
Bylund applied mathematical modelling to LC-MS peak purity determinations
using both real and simulated data [IS]. Bryant [16] compared electrospray
ionization (ESI) LC-MS and LC-MS-MS with DAD for examination of co-eluting
impurities in famciclovir and ropinirole. Tlus study demonstrated that for an
impurity set with similar W spectra, MS could be optimized in such a way to
consistently detect semi-coeluting known impurities at 0.1% whereas DAD could
not. All examined compounds contained m i n e groups, which typically provide
good sensitivity by ESI [17]. Salau [18] determined HPLC peak purity of
pesticide mixtures by thermospray MS. This study demonstrated the suppression
effects of exactly coeluting compounds in MS and that mathematical modelling
can detect coeluting compounds at the 5% level. This high detection limit is
attributed to the fact that thermospray exhibits greater noise and lower sensitivity
relative to other ionization techniques such as atmospheric pressure ionization
(API).
Fisher [19] examined the use of API LC-MS and LC-MS-MS techniques
to be used as chromatographic peak purity tools using one drug coeluted with four
impurities at levels ranging from 0.05% to 5.0%. Data indicated that, despite
spectral comparison of the pure compound with that of the spiked samples,
unambiguous detection of coeluting impurities was possible only at the 0.4%
level, falling short of the mandated level of 0.1%. The application of MS as a peak
purity tool for additional separation techniques was also discussed by Fanali [20].
A preliminary study examined ESI- MS as a tool for peak identification, peak
purity testing and selective monitoring of overlapping peaks with capillary
electrophoresis (CE). Other studies have interfaced MS with gas chromatography
(GC) to determine peak purity [21, 221. These GC and CE applications illustrate
the potential flexibility of MS as a general tool for peak purity. These studies
referenced above provide some evidence for the use of MS as a chromatographic
peak purity tool in specific instances. However, these studies do not provide an
evaluation of the general applicability of API LC-MS to detect low levels of
unknown impurities coeluting with a high level main analyte.
The impact of ionization technique (i.e. thermally assisted ESI and API)
was not sufficiently explored. As it is widely known that response factors vary
using mass spectrometric detection, it is important to test a diverse array of
compounds and impurities to provide a robust evaluation. This paper presents the
evaluation of API MS as a general tool for detection of coeluting unknown
impurities in HPLC. The investigation employed a single stage MS to facilitate
broad applicability. Data was obtained on an m y of drugs and impurities
coeluting at levels ranging from 10% down to 0.1%. The compounds tested
included acids, bases and zwitteriohs possessing a wide range of polarities,
spanning a mass range typical of pharmaceuticals and with varying ionizabilities.
Discussions of additional procedures and factors that can affect the application of
API MS to HPLC peak purity investigations are also discussed.
2.1.1 Impurities in drug substances
In general, impurities in drug substances are addressed from two perspectives:
The chemical aspects, which encompass classification and identification of
impurities, are in effect how to generate reports, set appropriate
specifications and describe analytical procedures.
Even more important are the safety aspects for the patients, who will be
using the final product when a new drug is brought to the market.
Comparative studies and genotoxicity testing are of increasing importance
in this context.
From the point of view of regulatory bodies, such as the US FDA, EMEA,
etc., impurities in drug substances are classified in these categories:
Organic impurities (process and drug related)
Inorganic impurities
Residual 'solvents
2.1.2 Case study
The five Application Notes in this compendium show a typical workflow
in an analytical method development and QAtQC laboratory during drug
development and commercialization, that is the analysis and determination of
production- related impurities in an active pharmaceutical mgredient. Analytical
procedures are essentially improved using Rapid Resolution LC by speeding up
the analysis and, combined with TOF, providing accurate mass information in the
initial phase of the investigation. There are multiple sources for organic impurities
at various concentration levels. They may arise most likely during the synthesis,
and storage of the active drug substance, but also during the manufacturing
process andlor storage of the final drug product and can come from:
Starting materials
By-products
r Intermediates
Degradation products
Reagents and ligands, but also from packing materials.
According to the FDA Guidelines "Impurities in Drug Substances",
identification of impurities below apparent levels of 0.1 % is generally not
considered necessary. However, identification should be attempted for those
potential impurities that are expected to be unusually potent, producing toxic or
pharmacologic effects at a level lower than 0.1 %. In all cases, impurities should
be qualified.
2.1.3 Analysis of by-products
The analytical procedures described in this document focus on the
discovery of starting materials, by-products and intermediates, and how they can
most effectively be determined early in the identification process. Further
information about the analysis of packaging material and organic volatile
impurities (OVI) can be found in the appendix. Impurity analysis follows the
typical method development workflow, method optimization / transfer and
subsequent routine use under cGMP conditions (Fig. 2. I).
Fig.2.1 Typical workflow in an analytical method development and
QAIQC laboratory
Regulatory authorities worldwide like ICH, USFDA, Canadian Drug and
Health Agency are emphasizing on the purity requirements and the identification
of impurities in Active Pharmaceutical Ingredients (APls). The process called
qualification of the impurities is acquiring and evaluating data that establishes
biological safety of an individual impurity; thus establishes biological safety of an
individual impurity; thus, emphasizing the need and scope of impurity profiling of
dmgs in pharmaceutical research. Impurities in drug substances and drug products
are classified as shown in Table 2.1.
Table 2.1 Classification and source of impurities in drug substance and drug products
Type of impurity Source of impurity
Process related: Dmg substance Starting material Intermediate By product Impurity in starting material Residual organic solvents Reagents, catalysts
Process related: Drug product Organic or inorganic Reagents Catalysts
Degradation: Drug substance or drug Organic degradation products- Product related substances
Degradation: Drug products Excipient interaction
Different official regulatory authorities have different definitions for
impurities: ICE defines irnpurities as by-products,' degradation products,
interaction products, intermediates, penultimate intermediates, related products
and transformation products. USP defines impurities as impurities in official
articles, ordinary impurities, and organic volatile impurities.
2.1.4 HPLC with 10 pm particle columns
. Although GC capillary columns were available in the early to mid 1970s,
these glass columns were difficult to work with and reactive. For these reasons in
the 1970s packed GC columns (as was used in HPLC) were commonly employed
for the analysis of seized drugs. For HPLC a major breakthrough occurred in 1972
with the popularization of silica based microparticulate packings [23] containing
totally porous 5 and 10 pn particles. In 1973 it became commercially feasible to
modify the silica surface via silanization and this led to the first 10 p reversed-
phase columns [24]. These columns contained a bonded non-polar moiety, which
in combination with a polar eluent separated solutes in order of their
hydrophobicities. It is interesting to note that HPLC using the 10 pm particle
packings and GC using these same packings (packed columns) gave comparable
separations for seized drugs [25, 261. A comparison of packed column GC and
HPLC for heroin analysis is shown in Fig.2.2 and 2.3.
Fig.2.2 Packed Column GC-FID chromatogram of heroin and related compounds. (Conditions: injection size 1.4 pL; column 6 ft x 114 in. (2 mm, id.) packed with 3% OV-1 on Chromosorb W-HP 801100 mesh. Temperature 235 OC)
Fig.2,3 HPLC chromatogram of heroin exhibit at sample concentration of 10 mglmL (Conditions: injection size 5 pL; column, 300 mm x 3.9mm 10 pm Porasil; 25% ammonia, methanol, chloroform (water washed) ( I t 200 +BOO); flow rate 2.0 mLlmin with W detection at 254 nm )
Ion pair chromatography and reversed-phase chromatography /27, 281
were important developments in the analysis of seized drugs, Using reversed-
phase chromatography it is preferable to analyze basic drugs in the un-ionized
state. This however, requires a mobile phase pH above 8. Unfortunately at such
high pH values modified silica particles become unstable and silica becomes
soluble. At lower pH value of 3.5, most basic drugs are ionized and thus have little
or no affinity for the non-polar bonded phase and instead interact with residual
silanol groups through ion exchange or adsorption mechanisms, Such interactions
can result in severe tailing. A possible solution to this problem is to form a
lipophillic ion pair complex with the salt of the basic drug using a counter ion
such as an alkyl sulfonate. This complex is far more retentive when used with a
non-polar bonded phase. The procedure also permits the concomitant detection of
acidic drugs for they will be neutral at such pH and also retained, To demonstrate
this, a 300 mm x 4 rnm 10 pm Bondapak C18 was applied to the analysis of a
wide variety of drugs of forensic interest including ergot alkaloids,
phenethylamines, opium alkaloids (heroin excluded), local anaesthetics,
barbiturates and other drugs of forensic interest [29]. The use of reversed. phase
ion pair chromatography allowed these compounds to be separated wing a single
mobile phase (40% methanol, 59% water, 1% acetic acid, 0.005 M
heptanesulfonic acid (pH 3.5). To further exploit the advantages of liquid phase
over gas phase separations the role of the stationary and mobile phases for
reversed- phase ion pair chromatographic spinition was investigated by the
author. Using Bondapak C18, Bondapak alkyl phenyl and Bondapak CN columns
and a mobile phase containing 1% acetic acid, a study was made of the effect of
counter ion size, counter ion concentmtion, water-methanol ratio and basicity on
the retention factor (k) and selectivity a values [30, 311. For a given stationary
phase and water-methanol ratio, the k values of the weakly acidic barbiturates and
an un-ionized base were fairly independent of counter-ion type and concentration.
For the ionized bases at a given mobile phase, the k values increased with
increasing size of counter-ion with both the C18 and alklyphenyl columns. For the
ionized bases on the C18 and alkylphenyl stationary phases and a given mobile
phase, the ratio of k values for any given set of counter-ions was fairly constant.
In general, higher variations in k with size of counter-ions were observed on the
C18 stationary phase than the alkyl phenyl stationary phase. These k ratios were
independent the size of the solute, but varied with the charge of the base. The
variation of k values with counter ion size on the C18 and alkylphenyl columns
was fairly independent of the methanovwater ratio employed. For the C18 and
alkylphenyl columns, the k of ionized bases increased with an increase in counter-
ion concentration especially for the more hydrophobic counter-ion ions. For the
CN column, no significant variation of k with counter-ion size or concentration
was observed for any of these basic drugs.
For similar solutes which differed in aliphatic character, increasing the
amount of water at a given counter ion (0.005 M concentration) increased values.
For the C18 and alkylphenyl stationary phases, increasing the size of the
alkylsulfonate counter-ion in mixtures of acids and ionized bases selectively
increased the retention of the bases. Although the values of ionized basic
compounds invariably changed with counter-ion ion size, there was no
generalized trend other that that the highest values were usually obtained using the
least hydrophobic counter ion. In most instances, the values of bases were
invariant to changes in counter ion concentration. Although for most solutes the
retention order was C18 > alkylphenyl > cyano at a given mobile phase
conditions, the C18 phase was preferred. The C18 phase was found to exhibit
greater overall selectivity than the other phases.
Based on these studies a wide variety of drugs of forensic interest could be
analyzed with superior resolution and speed over previously reported
methodology using a C18 stationary phase by replacing the heptanesulfonate
counter ion with methanesulfonate and using two isocratic mobile phases at
different methanol concentrations. [32]. None of these systems was satisfactory
for the separation of the major constituents in heroin. Therefore a third mobile
phase containing phosphate buffer, methanesulfonate and acetonitrile (pH 2.2)
was developed for the analysis of heroin samples [33]. Phosphate buffer was used
in place of acetate buffer because of its lower UV cut-off. For all three mobile
phases the use of a relatively high methanesulfonate concentration (0.02 M)
diminished the variation of retention time with concentrdtion of bases in the
working sample concentration range of 0 to 1.0 mg/mL.
The analysis of PCP on parsley or mint leaves was not possible using the
methanesulfonate counter ion, because of interference from compounds present in
the plant material. By using the lipophillic counter-ion ion dodecyl sulfate at a
concentration of 0.02 M, it was possible to manipulate retention so that the acidic
and neutral compounds related to the plant material eluted first, followed by the
basic compound PCP [34]. A major advance in mobile phase optimization
occurred in the 1980 when Glajch [35] outlined an approach for rational method
development for reversed- phase chromatography, based on Snyder's solvent
selectivity triangle [36] and a response surface as described by Snee [37]. This
computerized approach for optimizing selectivity, named by the authors
"overlapping resolution mapping", included keeping the solvent strength constant,
while changing the ty-pe(s) and amount(s) of organic modifier. Different solvents
such as methanol, acetonitrile and tetrahydrofuran have different abilities to
undergo hydrogen bonding, dipole interactions, dispersion, dielectric interactions
and electrostatic interactions. Overlapping resolution mapping was used for the
sepamtion of 26 fentanyl homologs and analogs using a 250 mm x 4.6 mm
Inertisil ODS3V column [38]. This approach predicted an optimum mobile phase
consisting of 81% phosphate buffer (pH 2.1), 4% methanol, lo0? acetonitrile and
5% tetmhydrofuraa
In 1975 a topological index 'tnolecular connectivity (X)" was introduced to
estimate solute cavity surface area [30]. Karger 1401 showed that hydrophobic
selectivity in HPLC can be accounted for by using x values. Using connectivity
indexes for the 26 fentanyl homologs and analogs, hydrophobic selectivity was
found to depend on the position of methylene substitution on the parent fentanyl
molecule and type of substituent [41]. It was M e r found that hydrophobic
selectivity was approximately independent of mobile phase composition for the
solvent mixture used in the overlapping resolution mapping scheme. Similarly,
hydrophobic selectivity was also found to be almost identical on both a 10 pm
particle silica-based 250 mm x 4.6 mm lnertisil ODs-3V column and a 10 pn
particle polymer based 150 mm x 4.6 mm RPI column (under normalized time
conditions). The silica based column exhibited significantly greater efficiency per
column length than the latter column.
2.1.5 HPLC with 3 and 5 pm particle columns
For GC a major breakthrough occurred in 1979 with the introduction of
fused silica capillaries [42]. These columns have much thinner walls than glass
capillary columns and the polyimide coating imparts mechanical strength. As such
they are flexible and have low reactivity. It is not surprising given the relative ease
of use and the significantly greater efficiencies and peak capacities that capillary
GC became popular in the 1980s. The use of capillary GC for heroin analysis is
shown in Fig.2.4. The peak emciencies of HPLC also improved in the late 1970s
and early 1980s with the advent of the smaller 3 and the increased use of 5 pm
particle size columns. Although these columns significantly increased
performance over the 10 pm particle columns, the GC capillary columns still
offered significantly greater peak capacity.
Fig.2.4 Capillary GC-FID chromatogram of major impurities, adulterants and diluents in heroin(Conditi0ns: column 25 m x 0.32 mm, fused silica (0.2 pm) SE-54. Temperature programmed from 150 OC a t 9 OC min -1 to 280°C (held for 0.5 min). Peaks: 1 = nicotinamide-TMS; 2 = acetaminophen- TMS; 3 = meconin; 4 = caffeine; 5 = glucose-TMS; 6 = phenobarbital-TMS; 7 = methaqualone; 8 = N-phenylnapthylamine; 9 = tetracosane (internal standard); 10 = acetylcodeine; 11 = acetylthebaol; 12 = morphine-TMS; 13 = 06-monoacetylmorphine-TMS; 14 = heroin; 15 = papaverine; 16 = phenolphthalein; 17 = noscapine 1431 )
Lurie and Carr conducted a comparison of 3 and 5 and 10 l m particle size
reversed phase columns for the separation of drugs of forensic interest [44]. Due
to the similar selectivity of the stationary phases and the higher efficiencies
obtained using 5 pm particles when compared to 10 pm particles, separations on a
125 mm x 4.6 mm 5 pn particle column were performed in at least half the time
compared to using the 250 mm x 4.6 mm 10 pm C18 particle columns. Rapid
analysis could be obtained using even shorter 3 p particle columns. However,
low plate counts and asymmetrical peaks were observed for basic drugs using the
100 mm x 4.6 mm HS/3 C18 3 pn particle column due to a large number of
unbonded silanol sites present on these particles. For the HS15 C 18 5 p particle
column M e r reductions in retention times and different selectivities were
obtained when the m i n e modifier hexylamine was added to the mobile phase
containing phosphate buffer pH 2.2. An approximately 9 fold decrease in retention
time could be obtained for the separation of heroin and its major by-products over
an earlier separation (10 p particles and no modifier) by using a combination of
smaller particle sized columns and an m i n e additive. Even after the addition of
hexylamine to the mobile phase to block unbonded silanol groups, the separation
of basic drugs on the 3 pm column was still inadequate. Using the HSl5 CIS 5 pm
particle column a separation of heroin and its major by-products, plus additional
by-products as well as major adulterants, was developed by studyiig the effect of
mobile phase parameters including amine concentration, organic modifier type
and eluent pH [45] (Fig.2.5). Since a lower solvent strength was necessary for
early eluting compounds and a higher solvent strength was needed for the more
hydrophobic adulterants, gradient elution was used. For the separation of acidic
and neutral acetylated rearrangement products of opium alkaloids, Lurie and Allen
used two 30 mm x 4.6 rnm 3 pm HSl3 C18 columns in series [46]. A separation
was developed based on the solvent optimization triangle.
Lurie used a 125 mm x 4.6 mm HS15 C18 5 p particle column for
analysis of manufacturing by-products in seized cocaine exhibits. Similar as in the
previous separation for PCP on plant material, 0.02 M dodecylsulfate was used in
order to control retention so that the retention order was acids, mono-protic
mines and di-protic mines. The chromatogram consisted first of carboxylic
acids such as benzoic acid, cinnamic acid (cis and trans) and several isomers of
truxillic and truxinic acids; next the mono-protic amines benzoylecgonine, cocaine
and cinnamoylcocaine (cis and trans) and finally isomers of the di-protic amine
truxilline. Lurie wed a 110 mm x 4.7 mm Inertisil ODs-3, 5 pm particle column
with isocratic conditions for the separation of benzodiazepines [47]. Anabolic
steroids were separated using a 250 mm x 4.6 mm Inertisil 5 p particle column
and a methanol water gradient [48].
Fig.2.5 HPLC chromatogram of major impurities and adulterants in heroin (Conditions: injection size 50 pL; column, 110 mm x 4.7mm 5 pm Inertisil ODs-3. Initial conditions: 5% methanol, 95% phosphate buffer (0.023M hexylamine, pH 2.2). Final conditions: 30% methanol, 70% phosphate buffer (0.023M hexylamine, pH 2.2), 20 minute linear gradient. Hold final conditions for 8 minutes; flow rate 1.5 mLImin)
2.1.6 HPLC with multiple detectors
The use of smaller size columns and mobile phase optimization has been
discussed in this chapter as means of increasing HPLC performance. Another way
of increasing this technique's performance is to increase the specificity of
detection. This is important in both qualitative and quantitative analysis. For
HPLC the relatively low peak capacity means that the reliability of compound
identification by retention time only (even for screening purposes) is poor. Baker
[49] showed that for 78 drugs separated by reversed- phase HPLC using a 300 mm
x 4 mrn 10 pm Bondapak C18 column, only 9% could be uniquely identified by
relative retention time alone. However, using relative retention time and 254:280
nm absorbance ratios (peak area or peak height ratios) and 95% of the drugs could
be uniquely identified.
Absorbance ratios will also provide information on peak purity which is
immensely important for quantitative analysis. Multi-wavelength detection also
allows more accurate and selective detection by using wavelengths close to the
absorbance maximum of a given solute. Other means of increasing specificity of
detection include fluorescence (FL), electrochemical (EC) and mass spectrometric
(MS) detection. The key to using these detection schemes with HPLC was the
commercial development of the thin-layer amperometric detector in the 1970 [SO]
the diode array (PDA) W detector in 1976 [51] and the thermospray (TSP)
interface in 1984. [52] Lurie used 220:254 nm absorbance ratios, as well as
relative retention times, for the screening of by-products and adulterants in heroin
samples. These ratios were also used to determine the peak purity of heroin.
Absorbance ratios of 215:230 mn were used, along with relative retention times,
to distinguish between 27 homologs and analogs of fentanyl. Lurie and Allen
employed dual UV detection, a programmable fluorescence detection and
electrochemical detection for the sensitive and selective detection of acidic and
neutral acetylated rearrangement products of opium alkaloids. For these solutes
higher UV wavelengths gave greater specificity, but not necessarily greater
sensitivity of detection. Although many of these compounds fluoresce,
fluorescence detection is more specific at trace levels because of its significantly
greater sensitivity for a smaller proportion of solutes, In contrast, for
electrochemical detection in the oxidation mode (0.85 V), only a relatively small
percentage of compounds will be electrochemically active with a sensitivity
approaching UV detection. Diode array (PDA) UV detection with the ability to
generate UV spectra "on the fly" offers significantly greater specificity for
qualitative analysis that the use of absorbance ratios. In addition this detection
mode allows W spectra to be generated across the peak to ascertain peak purity.
Lurie and McGuinness used relative retention times and PDA detection in
series with dual electrochemical detection to screen for the presence of by-
products and adulterants in heroin exhibits. For dual electrochemical detection in
the parallel mode, relatively high oxidation potentials of 1.0 and 1 . I V were used.
Response ratios were reported as one of three broad categories in order to
minimize changes in ratios which result from drifts in the reference redox
potential, sample over potential effects, or changes in temperature. Peak area
responses normalized to an internal standard nalorphine were used in order to
minimize effects due to electrode pacification. Library search sofhhrm was
developed for searching UV spectrum g e n d by the PDA detector and applied
for various applications including the identification of by-products and adulterants
in heroin samples 1531. Apex spectra at a measured retention time were searched
versus a library containing standard compounds. For a peak containing an impure
spectrum the upslope spectrum was successfully used to identify the peak.
Cocaine exhibits, Dual UV detection at 2 15 and 277 nm was employed. The lower
wavelength was used to measure all solutes except compounds that have a
cinnamoyl moiety. The higher wavelength allows for the sensitive and more
selktive detection of the latter compounds. The acquisition of UV spectra greatly
facilitated the determination of peak identity and purity. For the separation of
twenty benzodiazepines there was extensive overlap in retention times using UV
detection at 230 nm. However, all compounds gave unique UV spectra via PDA
detection. Three of the benzodiazepines (not well separated) were
electrochemically active in the oxidation mode and could be fkther identified by
employing PDA detection in series with dual electrochemical detection in the
parallel mode at 1.0 and 1.1 V. The benzodiazepines were also analyzed
separately using TSP-MS detection. Significantly enhanced specificity of analysis
over retention time alone was obtained since all solutes except clorazepate gave
[M+H]+ ions as the base peak. For the latter peak the base peak is [M + H -C02 - H20)+ ion. For most benzodiazepines, additional specificity is imparted to the
TSP mass spectra due to the presence of either a single C1 or Br atom or two C1
atoms, which give rise to ions due to natural isotopic abundances. Single ion
monitoring can be used to deconvolute the overlapping peaks. Lurie employed
PDA detection to enhance the specificity and sensitivity of analysis of anabolic
steroids. Although some of these solutes have identical UV spectra, for the few
unresolved peaks the UV spectra of the pair of anabolic steroids are unique. For
most steroid preparations only one of such a pair of compounds would be present.
The sensitivity k d specificity of quantitative analysis was enhanced by using
multiple wavelength detection at 210,240 and 280 nm.
In order to enhance the specificity of electrochemical, PDA or TSP-MS
detection for certain compounds, postelution photo irradiation was investigated
[54, 551. Continuous on-line post-elution photo irradiation wing a W lamp is a
means of converting eluting compounds into one or more photoproduct(s) prior to
detection. Continuous post-column photolytic derivatization using dual
electrochemical detection at 0.75 and 1.1 V facilitated the identification of cocaine
and selected adulterants, several of which had no response with the lamp off.
Cocaine under lamp-on conditions exhibited significantly greater specificity using
electrochemical versus UV detection.
Although the use of PDA detectors considerably increases selectivity by
providing UV spectra, UV spectra are not generally considered tools for absolute
peak identification due to the fact that compounds with similar structure may
produce identical spectra. As an alternative, TSP-MS can also significantly
increase selectivity by providing molecular weight information. However, usually
little or no additional information beyond molecular weight is obtained.
Additionally, certain compounds are not detected using TSP-MS. These
limitations can be overcome using post-elution irradiation. For example,
structurally related pairs morphine and 06- monoactetyl morphine and MDA and
MDMA each give similar UV spectra under lamp off conditions, but are easily
distinguished when the lamp is turned on. When using TSP-MS under lamp off
conditions, the structurally related pairs 03-monoacetylmorphine and 06-
monoacetylmorphine, and cannabidiol and A9-THC each give only identical rnlz
328 and m/z 3 15 (M + H)+ ions, respectively. However, under lamp on conditions
the thermospray mass spectra consisting of multiple ions, each are vastly different.
Barbiturates give no detectable TSP mass spectra under lamp-off conditions, but
multiple-ion mass spectra after post-elution irradiation.
2.1.7 Supercritical Fluid Chromatography
In 1982 Novotny and Lee 1561 reported on a workable instrument for
capillary supercritical fluid chromatography (SFC), which led to the first
commercial instrument in 1985. Because the diffusion coefficient of solutes in
supercritical fluids is about ten times greater than that in liquids (approximately
10000 times less than in gases) [57], capillary SFC could provide better resolution
and faster analysis than HPLC. In addition capillary SFC, which operates at lower
temperatures than GC, would be more favourable for the analysis of t h d l y
labile compounds. Next compared to GC capillary SFC would be more favourable
for the analysis of relatively non-volatile solutes. Finally, capillary SFC using
pure carbon dioxide as the mobile phase is compatible with the universal flame
ionization detector. Therefore it was not surprising that capillary SFC generated
much interest and excitement as a possible alternative technique to HPLC.
Lurie in 1988 was the first to demonstrate a multidimensional approach
coupling HPLC with capillary SFC [58], a schematic of which is shown in Fig.
2.6. The relatively non-volatile high molecular weight isomeric millines (eleven
possible isomers of molecular weight 658), eluted with methylene chloride, were
he* cut from an HPLC size separation column onto a capillary SFC system.
Unfortunately, the partial separation obtained for these solutes using SFC was not
better than could be obtained using HPLC.
Fig.2.6 Schematic diagram of coupled HPLC- capillary SFC system [From
reference 581
2.2 SEPARATION AND QUANTIFICATION OF IMPURITIES:
CONCEPTS OF UHPLC
Among impurity profiling tools, the separation technique such as HPLC,
TLC and GC are the most preferred. Even though thin layer chromatography has
been used extensively in the past, cunently pharmaceutical analysis adores HPLC
as its core separation tool. It is not only the inherent quality that comes with
HPLC but it is its accuracy, fine tuned results and hyphenation with other
detectors that made it the most versatile technique among separation methods. Gas
chromatography, even though an ~ ~ ~ u r a t e method owing to its destructive nature
it has been always considered as an alternative technique. It is applicable for
special impurities estimations like residual solvents in activate pharmaceutical
ingredients and formulations. Capillary electrophoresis (CE) and supercritical
fluid chromatography (SFC) are the other promising separation technologies that
gave an edge to separation science.
Since its discovery, HPLC has undergone sea changes with respect to its
technical capabilities. In this direction the latest development has given rise to an
improved version called Ultra High Performance Liquid Chromatography
(UHPLC). The whole idea of introducing the UHPLC systems is to reduce
analysis time considerably without compromising on performance. UHPLC
increases throughput as well as decreased flow rates. Some of the worth
mentioning features of the individual modules of an UHPLC and their
characteristics are as follow.
2.2.1 The,auto samplers
The present day auto samplers can deliver with high precision submicrolitre
volumes (0.1 pl to 1500p1) to the flow at any mobile phase flow rate and pressure.
They possess the zero cany over property, which is highly desirable for impurity
profiling studies.
2.2.2 Photodiode array detector (PDA)
The PDA detector plays a crucial role in the identification of compounds and
determination of peak purity. The diode array detector can acquire data from 190-
950 nm with a speed and frequency of SoHz for both spectra and signals. Up to
eight signals at different wavelengths can be acquired apart from recording high-
resolution UV spectra with a wavelength accuracy of i 1 nm.
2.23 The Pump
The pumps deliver flow rates of 0.05 mVmin to 5 ml/min. The high-
pressure tolerance limit ranges fieom 600 ban to 1500 bar in some systems.
Column thermostat will have a temperature range of 0°C to 100aC with
tempaature stability of < M.OS°C. Apart from the above mentioned advakced
characteristics in various modules of an UHPLC, an another aspect that needs to
be mentioned here is its hyphenation and compatibility with various other
detectors. The detectors that can be conveniently connected to monitor the eluent
of an HPLC are:
Mass Spectrometers (LC-MS) (LC-MS-MSn)
Nuclear Magnetic resonance spectrometer (LC-NMR)
Others detectors include LC-ELSD, LC-CLND, LC-PDA etc.
2.2.4 Column the heart of HPLC
The aim of HPLC analysis is to separate efficiently the closely related
individual components of a mixture to a best possible level and within a shortest
possible time with a high degree of accuracy and repeatability. Even though
various functions of an HPLC are controlled and dictated by individual modules
of an HPLC, column is responsible for the separation. It is the stationary phase
(Packing material) in combination with the mobile phase which is responsible for
the separation. There are varieties of column stationary phases that are available
as commercial products. To name some are C I R , ~ ~ , C4, Cyano (CN), Phenyl etc.
In general in most of the cases it is the silica that is used as a stationary support for
the above mentioned functionalities which are bonded covalently to the oxygen of
Si-0. The most widely used column technology for the analysis of majority of
small molecule pharmaceuticals is the Cls phase. Keeping the other things
constant the most important parameter that has a direct bearing on separation is
the particle size of the stationary phase. The relation between the resolution, plate
number, retention and resolution are given by the formula,
Thus, resolution (Rs) increases with increase in column length (L) or decrease in
particle size (dp). Even though increase in column length increases resolution, it
increases retention proportionately, thus increasing the analysis time multiple
folds. Hence the best choice is to decrease the particle size to increase resolution.
This idea has constantly evolved giving b i to the sub 2 pm particle columns.
The history of development of column stationary phase particle technology is
mentioned in Table 2.2. However, the decrease in particle size of the stationary
phase exerts high resistance to the flow, leadiig to increase in column pressure.
To tackle this problem the HPLC system has been redesigned to operate and
withstand high pressures. This concept has given birth to the ultra high
performance liquid chromatographic system (UHPLC).
The advanced technical features in these systems are:
(A)The upper pressure limits range from 600 to 1500 bar. As against 400 bar of
the traditional HPLC system
(B) High frequency data acquisition detector system (typically 80Hz)
Thus by increasing the pressure tolerance, columns with particles as small as
1.8 pm can be used. Various particle sizes and their nominal pressures are
summarized in the following table with column and mobile phase conditions:
100~4.6 rnm, 1 .OmVmin, 5050 (vlv) water: Acetonitrile (Table 2.3).
Table 2.2
History of commercial HPLC column particle development and tbeir efficiency
Year of Nominal size Approximate
Acceptance plates11 5cm
1950s 100pm Irregular shaped 200
1967 50pm Glass beed - pellicular 1000
1972 1 Opm 6000
1985 5 ~ m 12000
1992 3 - 3.5 pm 22000
1998* 1.5 pm* non - porous 30000
1999 . 5.0 pm pellicular 8000**
2007/2008 2.7 pm pellicular 32000***
+non porous silica or resins
**300 A' pore for protein
***90-120 A' Pore
Table 2.3
Various particle size columns and their back pressure for a given mobii phase and flow rate condition
Particle size Pressure Theoretical
Psi bar Plates (N)
As the chromatographic run time decreases with increase in performance of the
column, the peak widths also will decrease considerably. The decreased peak
widths may lead to loss of sensitivity. Hence, the data acquisition rate has to be
increased to keep in phase with decreased peak widths. To address this issue
detector with high efficiency data acquisition (8OHz) was introduced.
2.2.5 Preparative HPLC
Preparative HPLC is the most efficient and convenient tool for the
purification of wide varieties of pharmaceutical, chemical, biological and plant
extraction products. Preparative HPLC derives its high efficiency from its highly
sophisticated instrumentation as well as efficient column technology. In the
pharmaceutical industry, scientists o h come across compounds closely related
to each other such as related substances, degradation products, chiral impurities
(enantiomen) etc., which need to be isolated and characterized in their pure form.
Most of these separations cannot be achieved by traditional column
chromatography or crystallization techniques. Thus, the Preparative HPLC in
combination with highly efficient preparative HPLC columns is the solution for
the difficult separations of related substances.
2 3 SPECTROSCOPIC TOOLS FOR THE STRUCTURE ELUCIDATION
OF UNKNOWN IMPURITIES1 DEGRADANTS: HRMS AND NMR
2.3.1 High Resolution Mass Spectrometry (HRMS)
Mass spectrometry is a powerful analytical tool for the analysis of molecule,
both small and large. It can be used to identify unknown compounds, quantify
known compounds, and obtain information about chemical structure of both small
and large biological molecules. The ability of mass spectrometry to do all of this
from minute amounts of substances, often less than a billionth of a gram makes it
a highly useful technique in both established and emerging branches of science.
The versatility of this technology lies in its sensitivity and applicability to large
bio-molecules such as proteins, peptides etc. to small organic molecules.
The world's first commercial mass spectrometer was launched in 1948
which made use of electron impact ionization (El) with a mass range of 300 amu
and limited resolution. During 1950s time-of flight and quadruple analysis were
conceived. The next major development was of gas chromatography and its
coupling with mass spectrometry. This is not only allowed the first time direct
analysis of mixture of analytes but was also a trigger for the development of
present day mass spectrometry. For the past 30 years new ionization techniques
like fast particle desorption, electrospray ionization and matrix-assisted laser
desorptionlionization were discovered, The ionization techniques were invented
and developed in order to analyse complex molecules in their intact native form
and to aid hyphenation with other separation techniques like HPLC.
Combination of two analytical instruments yields great amount of
analytical information simultaneously and rapidly. Most of the current technical
advancements in analytical instrumentation are aimed at hyphenation. Direct
coupling of Gas spectrometer with MS was initial step towards hyphenation. The
gaseous molecules need to be ionized before they are fed in to the mass
spectrometer. Ionization is a very crucial process in mass spectrometry.
Generation of ions instantaneously h m neutral molecules is what happens inside
an ionization source. Electron impact ionization utilizes fast moving electrons to
knockout electrons from neutral molecules forming ions which are sucked in to
the mass spectrometer by means of electrometer by means of electrostatic
attractions and vacuum. This process is harsh and mainly gives fragments of the
molecular ion peak. This is the technique still vastly used in GC- MS systems
apM from chemical ionization. These techniques cannot be applied to the LC-MS
hyphenation where the eluent is a liquid. For this purpose electro-spray ionization
was invented and put in to use in early 1980s. In this technique highly charged
liquid droplets dispersed fiom a capillary in a high electric field are evaporated
spontaneously by hot nebuliser gas such as nitrogen. The free charged particles
formed in this process are attracted towards the MS inlet by electrostatic fields.
There are varieties of mass analysers available today with varied
analytical capabilities such as mass range and resolution. The most basic analysers
will be the quadruple mass spectrometers. Followed by TOF and other mass
analysers such as ion traps, ion cyclotron resonance MS etc. Single quadruple
mass spectrometers are the most vastly used machines in today's world for both
qualitative and quantitative analysis. Single quadruple mass spectrometric
experiments yield mass to charge ration of an ion rounded to its nearest whole
number. High resolution mass spectrometers such as time-of-flight and double
focusing FT-ICR mass spectrometers are capable of measuring the exact mass of
an ion. This is useful for interpretation because each element has a slightly
different mass defect. This "mass defect" is the difference between the mass of the
isotope and the nominal mass (which is equivalent to the number of protons and
neutrons). The atomic mass scale is defined by carbon-12 with a mass of exactly
12.0000 u (Table 2.4). The exact mass of a specific isotope is determined relative
to 12c by high resolution mass spectrometry. High resolution mass spectrometry
can distinguish compounds with the same nominal mass but different exact mass
caused by different elemental composition.
Molecular formula of molecular ion can be determined directly by
comparison of ion mass at high resolution with possible compositions using
accurate masses of individual isotopes. Thus the exact mass calculated fiom the
54
following table gives a value of 28.03 130 for ethane. When the mass is measured
by HRMS and if it yields a mass value 28.03130 it not only conforms ethane
molecular formula but also distinguishes from other two compounds yielding
similar molecular weights such as N2 and CO (Table 2.5).
Table 2.4
Exact masses of different elements
Table 2.5
Identification of compounds by HRMS
Molecule Exact Mass
Atom
Exact Mass
Table 2.6
I2c
12.000000
'H
1.007825
Identification of compounds by HRMS
Molecule Exact Mass
CH20 30.01056
The nominal mass of CH20, C2Ha and NO is 30 amu. The ordinary mass
spectrometer cannot distinguish them as all three yields the same mass of 30 m u
6~
15.99491
1
14.00307
whereas, the HRMS can distinguish them according to their exact masses as
mentioned in the table 2.6. Thus applying the above. masses to calculate exact
mass of any unknown molecular formula can be predicated. Calibration is crucial
for HRMS instrumentation.
2.3.2 Nuclear Magnetic Resonance P M R )
Nuclear magnetic resonance (NMR) spectroscopy is a powerfil and
versatile spectroscopic technique for the investigation and understanding of
unknown molecular structures and dynamics. Apart from structure elucidation
NMR is finding applicability in other pharmaceutical analysis also. This technique
is applicable to both liquid and solid samples. It is an extremely useful technique
as it is anon destructive analytical tool. NMR was developed in the year 1946 and
over the next 50 years NMR evolved in to premier organic spectroscopic
technique.
NMR spectroscopy makes use of the magnetic properties of nuclei inside the
atoms to yield chemical information of each atom in a molecule. According to
quantum mechanics, magnetic momentum of the nuclei arises from a net spin of
the sub atomic particle (electrons, protons and neutrons). In some atoms, the spins I2 16 32 are paired up ( C, 0, S) and some nuclei posses over all spin
( 1 ~ , 1 3 ~ , 1 9 ~ , 1 5 ~ , 3 1 ~ ) . If both the number of neutrons and the number of protons are
even, the nucleus has no spin. If the number of neutrons plus the number of
protons is odd, then the nucleus has a half-integer spin (i.e. 112, 312, and 512). If
the number of neutrons and the number of protons are both odd, then the nucleus
has an integer spin (i.e. l , 2 and 3).
In mechanical terms, the nuclear magnetic moment of a
nucleus will align with an externally applied magnetic field of strength Bo in only
2It1 ways, either with or against the applied field Bo. For a nucleus with spin = %
and a positive gyromagnetic ratio (y) only one transition is possible with a lower
energy level a (spin = + %) (parallel) and a higher energy level
(antiparellel)(spin =-1/2). The rotational axis cannot be exactly parallel or
antiparellel but will presses at an angle with angular velocity
o, is also called Larmor frequency
Each nuclei has a characteristic value of y, related to magnetic moment p
and spin number I
This precession process generates an electric field with frequency o, and if
we irradiate the sample with radio waves (in the MHz frequency range) the proton
will absorb energy and is promoted to the higher energy state. This energy
absorption is called resonance because the frequency of the applied radiation and
the precession coincide or resonate.
With increase in external magnetic field strength (BO), resonance frequency of
a given nuclei also increases (Table 2.7). Thus for different nuclei with different
gyromagnetic ratio (y) different frequencies must be applied to achieve resonates
at 400MHz and carbon at 100MHz.
Table 2.7
Magnetic field strengths and corresponding proton resonance frequencies
BO (Tesla) 'H Frequency (MHz)
1 1.75 500
18.8 800
21.2 900
23.5 lo00
Since the energy changes involved in NMR spectroscopy is very small (radio
fkquency), the signal sensitivity becomes a problem. In Fourier transformation-
NMR all spectral width are irradiated simultaneously with a single radio
hquency pulse. Following imadiation with radio frequency, the nuclei magnetic
moments press away h m applied magnetic field. Once the application of RF is
withdrawn, the magnetization will undergo 'relaxation' process emitting the signal
called 'free induction decay' (FID). FID is a time domain signal, which on Fourier
transformation gives hquency domain signal. This whole process is called a
'scan', which is then repeated several times to improve signal-to-noise ratio.
2.3.2.1 Chemical Shift
Now if all the protons and carbons in a molecule resonate at a single
frequency a single peak would have been obtained, giving no information about
individual protons or carbons. Rut practically the proton and carbon signals from a
molecule are generally spread over a frequency range. This is called the chemical
shift which is measured in terms of ppm scale with reference to TMS
(tetramethylsilane) signal. The chemical shift of individual proton with respect to
TMS indicates the chemical nature of that proton. Chemical shift indicates the
nature of hybridization of carbon, the kind of atom it is bonded to for ex. Oxygen,
halogen, another carbon etc.
Chemically different hydrogen's in an organic molecule do not experience
the same magnetic field. Electrons shield the nucleus thereby reducing the
effective magnetic field and requiring energy of a lower frequency to cause
resonance. On the other hand, when electrons are withdrawn from a nucleus, the
nucleus is de-shielded and feels a stronger magnetic field requiring more energy
(higher frequency) to cause resonance. Thus, NMR can provide information about
an atoms electronic environment.
2.3.2.2 umber of different hydrogens
The area under the NMR resonance is proportional to the number of
hydrogens which that resonance represents. In this way, by measuring or
integrating the different NMR resonances, information regarding the relative
numbers of chemically distinct hydrogens can be found. Experimentally, the
integrals will appear as a line over the NMR spectrum. Integration only gives
information on the relative number of different hydrogen's, not the absolute
number.
23.23 Splitting or coupling
NMR not only gives the total number of protons or carbons but also provides
information on how many hydrogen neighbow exist for a particular hydrogen or
group of equivalent hydrogen's. In general, an NMR resonance will be split into
~ + l . Peaks where N=number of hydrogen's on the adjacent atom or atoms. If
there are no hydrogen's on the adjacent atoms, then the resonance will remain a
single peak, called a singlet. If there is hydrogen on the adjacent carbon atom, the
proton resonance is split into two peaks of equal size, a doublet. Two hydrogen
atoms on the adjacent atoms will split the resonance into three peaks with an area
in the ratio of 1:2:1, a triplet. If there are 3 hydrogen atoms on the adjacent atoms,
the resonance will be split into four peaks with an area in the ratio of 1:3:3:1,
called a quartet. The split distances in terms of hertz are called coupling constant
and is also an indication of the nature and intensity of coupling between protons.
Thus the one dimensional proton and carbon experiments will give
information about the number, their chemical nature and to some extent their
position within the molecule. Additional ID experiments of carbon called DEPT
(Distortion less enhancement by polarization transfer), APT (Attached proton test)
indicate the number of protons directly bond to the carbon atom,
2.3.2.4 Nuclear Overhauser Effect (NOE)
An interesting phenomenon that can be studied in NMR spectroscopy is
through space interaction of protons within a molecule. Saturating the electron
magnetic resonance intensity to be enhanced by a factor of the order of lo3 is
called Nuclear Overhauser Effect (NOE) and was first discovered by Albert
Overhauser in 1953. A proton which is spatially close to another proton by less
than 5 Angstroms will show the transfer of magnetization between each other
when saturated by irradiation. This experiment is carried out by constantly
irradiating one proton and any magnetization picked by other proton is measured.
Both 1D and 2D set of experiments are available named as NOESY experiments.
These experiments will be very useful to confirm s t~~c tures in special cases as
exemplified in this research work.
23.2.5 Other 2D Experiments
The information obtained fiom ID NMR experiments may not be sufficient
to ascertain the structure of the molecule. There are varieties of 2D experiments
that are available to assign positions of different protons, carbons and nitrogen's
in a molecule. These are called 2D correlation experiments. Correlation between
similar atom types (proton-proton) are called homonuclear correlation
experiments (Ex: Cosy). Correlation between different atom types (proton-carbon
or proton-nitrogen) are called hetero-nuclear correlation experiments (Ex: HSQC,
HMBC). All these experiments provide wealth of information about the structure
of the molecule.
2.4 CHIRAL ANALYSIS O F PHARMACEUTICAL COMPOUNDS,
CHlRAL STATIONARY PHASES (CSP)
2.4.1 Chiral analysis of Pharmaceutical Compounds
Any two molecules, which are not super imposable, but are mirror images of
each other, are called chiral molecules. This terminology does not indicate any
aspect of chiral purity or chiral identity but just 1848 by Louis Pasteur, a French
chemist and biologist. For the first time he had separated by hand the two isomers
of sodium ammonium tartrate. Chirality is a hallmark of many molecules existing
in nature and many such molecules are unichiral in nature. For example chiral a-
amino acids, and the peptides and proteins containing them, sugars and their
polysaccharides, steroids, antibiotics and many other compounds from nature are
unichiral. Most of the biologically active molecules obtained from nature are
chiral, for ex. morphine, quinine etc.
Although they have the same chemical structure, most enantiomers of
racemic drugs exhibit marked differences in biological activities such 8s
pharmacology, toxicology, pharmacokinetics, metabolism etc. There are a number
of examples to state the importance of chirality in h g s and their physiological
effects. Thalidomide is a sedative drug that was prescribed to pregnant women,
from 1957 into the early 60s. It was marketed in at least 46 countries under
different brand names. When administered to pregnant women during the first
trimester of pregnancy, Thalidomide prevented the proper growth of the foetus,
resulting in horrific birth defects in thousands of children around the world. The
drug was found to cause teratogenicity and neuropathy. Because thalidomide
molecule is chiral and the molecule that administered was racemic. One c h i d
form was a sedative, whereas the other one was later found to cause foetal
abnormalities. The tragedy is claimed to have been entirely avoidable had the
physiological properties of the individual thalidomide enantiomers would have
been tested prior to commercialization.
Aspartame is a sweetening agent that is more than a hundred times sweeter
than sucrose. And yet, the other enantiomer molecule is bitter. (S)-carvone
possesses the odour perception of caraway while the other enantiomer (R)-corvine
has a spearmint odour.
Although enantiomers have the same chemical structure, most isomers of
chiral drugs exhibit marked differences in biological activities such as
pharmacology, toxicology, pharmacokinetics, metabolism etc. Some mechanisms
of these properties are also explained. The mechanism of interaction of the chiral
drug and the biological target is now being understood with the help of crystal
structures. Hence, it is very important to understand the role of individual
enantiomers at discovery stage. Thus helping to eliminate the unwanted
enantiomer at the clinical/discovery stage and select and enhance the activity and
reduce the side effects of the selected new chemical entity. This necessitates the
scientific community to enhance and acquire knowledge on enantio selective
synthesis and also equip with strong analytical skills to separate and quantitate the
enantiomers with speed and accuracy.
Among different ways of estimating enantiomers currently, HPLC is one
of the viable options. Different types of chiral stationary phases have been
developed for the separation of enantiomers. There are two types of c h i d liquid
chromatographic separation methods
i) D i t method
By direct diastereomer formation on stationary phase or by addition of
chiral selector in mobile phase
ii) Indirect method
By diastereomer formation by reaction with homochiral reagent
Direct separation method is now the preferred means of achieving c h i d
separation especially using chiral stationary phases whereas the use of chiral
selectors in mobile phases is an expensive process. Indirect method is now a thing
of past as it involves multiple reaction steps and may lead to racemisation during
this process. Thus use of high performance liquid chromatography (HPLC),
capillary electrophoresis (CE), Supercritical fluid chromatography (SFC) and gas
chromatography (GC) instrumentation with chiral stationary phases (CSPs) is the
current trend of chiral analysis both for analytical as well as preparative scale
separations. Different types of detectors have been employed to detect,
quantitative and identify the enantiomers after separation.
2.4.2 Chiral Stationary Phase (CSP)
Chiral Stationary Phase (CSP) is the heart of chiral separation. Various
types of CSPs have been developed and commercialized for the separation of the
racemic mixtures.
2.4.2.1 Macrocyclic glycopeptide antibiotics as CSPs
Macrocyclic glycopeptide antibiotics as CSPs were introduced by
Armstrong in 1994. The antibiotics used for chiral resolution are vancomycin,
vancomycin aglycon, teicoplanin, teicoplanin aglycon, ristocetin A, thistrepton,
rifamycin, fradiomycin, streptomycin, kanarnycin, and avoparcin. However, the
most commonly used antibiotics are vancomycin, teicoplanin, teicoplanin aglycon
and ristocetin A. All of these contain ionisable groups at different pH values in
their structures. Their molecular masses are between 1000 and 2100. The
glycopeptides are amphoteric, containing both ionisable acidic and basic groups.
Thus, they can be positively charged, negatively charged, or neutral depending on
the pH of the mobile phase.
Vancomycin, it is very effective for the enantiorecognition of anionic
compounds particularly those containing carboxylic acid group in their structure.
Its molecular weight of 1449 makes it the smallest of the three macrocyclic
glycopeptides. This selectivity is mainly due to the presence of m i n e groups in
the structure. There are 18 chiral centres in this molecule with 3 cavities. It has a
pendant, freely rotating, disaccharide moiety consisting of D-glucose and
vancosamine and an N-methyl amino acid side chain around three b e d
macrocyclic rings bridged by five aromatic rings linked by ether and peptide
bonds. Vancomycin has nine hydroxyl groups around the "basket shaped" aglycon
and on the attached disaccharide moiety, two mine groups (one primary and one
secondary), and one carboxylic acid group. There are five aromatic ring structures.
Hydrogen donor and acceptor sites are available close to the ring structure.
Several functional groups such as carboxylic, hydroxyl, amino, and amido are
mainly responsible for the ionization of these antibiotics in buffer with various
pHs and compositions and, therefore, it is enantioselective in nature.
Teicoplanin and teicoplanin aglycon: Teicoplanin is a macrocyclic
glycopeptide antibiotic that is structurally related to vancomycin and ristocetin A.
Teicoplanin exhibits a very slight anionic character even at acidic pH. There are
23 chiral centres in this molecule with four cavities. There are three sugar
moieties. Hydrogen donor and acceptor sites are available close to the ring
structures. It also contains a hydrophobic acyl side chain attached to a 2-amino-2-
deoxy-a D-glucopy~anosyl moiety, which activates its surface and enables the
formation of micellar aggregates. This structural feature is characteristic of
teicoplanin. These properties render teicoplanin highly stereo-specific in nature.
Teicoplanin without a sugar part is called teicoplanin aglycon antibiotic contains
almost all of the groups and cavities as in teicoplanin and therefore, has similar
chemical and physical properties. Teicoplanin aglycon does not show the
aggregational behaviour.
Ristocetin, it is the largest of all with a molecular weight of 2066. It has
the greatest number of stereogenic centres, i.e, 37. Ristocetin has structure very
similar to teicoplanin and vancomycin. An aglycon portion with four fused
macrocyclic rings, one tetramxhaxide moiety and six monosaccharides together
with 38 stemgenic centres characterizes it. It bas also seven aromatic rings, 21
63
hydroxyl groups, two primary amine groups, six arnido groups (amide linkages)
and one methyl ester. This adbiotic also contains three cavities.
2.42.2 Polysaccharide based chirnl stationary phases
Polysaccharides are the most abundant optically active natural polymers on
earth. These compounds themselves could not be used as commercial CSPs
because of their poor resolution capacity and problem in handling. The
polysaccharide polymers such as cellulose, amylase and chitin can be readily
derivatized to esters and carbarnates through reaction with acid chlorides and
isocyanates respectively. These polymer chains lie side by side in a linear fashion
in cellulose and in helical fashion in amylase. The CSPs based on polysaccharides
are some of the most popular and show a very high chiral resolving power and are
widely used for both analytical and preparative applications. About 20 derivatives
of amylase and cellulose are commercially available. The structures of some of
these CSPs used in this study are depicted in the following pages.
2.4.2.3 Pirkle-Type chiral stationary phases
The CSPs are broadly classified into two categories called 11-acceptor and
ll-donor phases. Whelk-01 and Whelk-02 are a "hybrid" type of this category,
which is a n-electron acceptor-donor CSP.
REFERENCES
1) International Confeoence on Harmonisation (ICH), Guidance Q3A,
Impurities in New Drug Substances, Federal Register, Jan. 4, 1996 (61 FR
371). (Available at http:/h.iEpma.org/ichl .html).
2) Food and Drug Administration (FDA) Center for Drug Evaluation and
Research(Avai1able at htlp:/lwww.fdagov/cder/guidance12452fnl.).
3) International Federation of Pharmaceutical Manufacturers Association
(Available at http://www.ifpma.org).
4) Lincoln, D., Fell, A.F. and Anderson, N.H., "Assessment of
Chromatographic Peak Purity of Drugs by Multivariate Analysis of Diode
Array and Mass Spectrometric Data", J. Pharmaceut. Biomed., 10, 10-12
(Oct-Dec 1992), pp.837-844.
5) Sharaf, M.A., "Assessment of chromatographic Peak Purity", Adv.
Chromatogr., 37(1997), pp. 1-28.
6) Castledine, J.B. and Fell, A.F., "Strategies for Peak Purity Assessment in
Liquid Chromatography", J. Pharmaceut. Biomed. Anal., 11,l (Jan. 1993),
pp.1-13.
7) Polster, J., Sauerwald, N., Feucht, W. and Treutter, D., "New methods for
spectrometric peak purity analysis in chromatography", J. Chromatogr., A,
800 (1998), p.121.
8) Fabre, H., Le Bris, A, and Blanchin, M.D., "Evaluation of different
techniques for peak purity assessment on a diode-array detector in liquid
chromatography", J. Chromatogr., A, 697 (1995), p.81.
9) Stewart, C.W., "The use of liquid chromatography-mass spectrometry to
demonstrate method specificity", American Laboratory, 32, 9, News
Edition (April 2000), p. 18.
10) Ermer, J. and Vogel, M., "Applications of hyphenated LC-MS techniques
in pharmaceutical analysis", Biomed. Chromatogr., 14,6 (2000), p.373.
11) Mistry, N., Isrnail, I.M., Smith, M.S., Nicholson, J.K., Lindon and J.C.,
"CCharacterisation of impurities in bulk drug batches of fluticasone
propionate using directly coupled HPLC-NMR spectxoscopy and
HPLCMS", J. Pharmaceut. Biomed. Anal., 16 (1997). p.697.
12) Lacroix, P.M., Dawson, B.A., Sears, R.W., Black, D.B., Cyr, T.D. and
Ethier, J.C., "Fenofibrate Raw Materials: HPLC Methods for Assay and
Purity and an NMR method Purity", J. Pharmaceut. Biomed. Anal., 18
(1998), pp.383-402.
13) Larson, A.A. and Dalo, N.L., "Quantification of Tryptamine in Brain
Using High Performance Liquid Chromatography", J. Chromatogr.
Biomed. Appl., 48(1), (Feb. 14, 1986), pp.37-47.
14) Zrybko, C.L. and Rosen, R.T., "Determination of Glucosinolates in
Mustard by High Performance Liquid Chromatography Electrospray Mass
Spectrometry", ACS Symposium Series., 660 (1 997), pp. 125-137.
15) Bylund, D., Danielsson, R. and Markides, K.E., "Peak purity assessment in
liquid chromatography-mass spectrometry", J. Chromatogr., A, 915
(2001), p.43.
16) Bryant, D.K., Kingswood, M.D. and Belenguer, A,, "Determination of
Liquid Chromatographic Peak Purity by Electrospray Ionization Mass
Spectrometry", J. Chromatogr., A, 72 1, 1 (Jan. 15, 1996), pp.4 1-45.
17) Willoughby, R., Sheehan, E. and Mitrovich, S., A Global View of LCIMS,
lS' edition, Global View Publishing, Pittsburgh, PA, 1998.
18) Salau, J.S., Honing, M, and Tauler, R., "Resolution and quantitative
determination of coeluted pesticide mixtures in liquid
chromatographythermospray mass spectrometry by multivariate curve
resolution", J. Chromatogr., A, 795, 1 (Jan. 30, 1998), p.3.
19) Fisher, D., Morgan, D., Moseley, M.A. and Whitlock, L.,
"Chromatographic peak purity determination using LCIMS: A practical
examination", Proceedings of 44th ASMS Conference on MS and Allied
Topics, Portland, Oregon (May 12- 16, 1996).
20) Fanali, S'., Desiderio, C., Schulte, G. and Heitmeier, S., "Chiral capillary
electrophoresis-electrospray mass spectrometry coupling using
vancomycin as chiral selector", J. Chromatogr., A, 800, 1 (1 998), p.69.
21) Krock, K.A., Ragunathan, N. and W i l k i , C.L., "Multi-dimensional gas
chromatography coupled with infra-red and mass spectrometry for analysis
ofeucalyptus essential oils", Anal. Chem., 66 (44) (Feb. 15, 1994), p.425.
22) Anon, ''Determination of the insecticide pirimicarb in cereals,"
Laborhaxis, 18 (7), (Jul. 1994), pp.34.
23) R.E.Majors, Anal. Chem., 44 (1972), 1722.
24) U.D.Neue, HPLC Columns: Theory, Technology and Practice, Wiley-
VCH, New York, 1997.
25) J.D.Wittwer, Forensic Sci. Int., 18 (1981), 2 15.
26) PP.DeZan and J. Fasanello, J. Chromatogr. Sci., 10 (1972), 333.
27) S.Eksborg, P.O.Lagerstrom, R.Modin and S.Schill, J. Chrornatogr., 83
(1973), 99.
28) J.A.Korpi, D.R.Wittwer, W.G.Haney, in: Meet. Fed. Anal. Chem.
Spectrosc. Soc., Indianapolis, 1975.
29) I.S.Lurie, J. Assoc, Off. Anal. Chem., 60 (1977), 1035.
30) 1.S.Lurie and S.Demchuk, J. Liq. Chromatogr., 4 (1 981), 337.
31) 1.S.Lurie and S.Demchuk, J. Liq. Chromatogr., 4 (1981), 357.
32) I.S.Lurie, J. Liq. Chromatogr., 4 (1981), 399.
33) I.S.Lurie, S.M.Sottolano, S.Blasof, J. Forensic Sci., 27 (1982) 519.
34) I.S.Lurie, Am. Lab., October 1980,36.
35) J.L.Glajch, J.J.Kirkland, K.M.Squire and J.M. Minor, J. Chromatogr., 199
(1 980), 57.
36) L.R.Snyder, J. Chromatogr. Sci., 16 (1978), 223.
37) R.D.Snee, Chemtech., 9 (1979), 702.
38) I.S.Lurie, A.C.Allen and H.J.Issaq, J. Liq. Chromatogr., 7 (1984), 463.
39) L.B.Kier, L.H.Hall, W,J.Murray and M.Randi, J. Pharm. Sci., 64 (1975),
1971.
40) B.L.Karger, J.R.Gant, A.Hartkopf and P.H. Weiner, J. Chromatogr., 128
(1 976) 65.
41) 1.S.Lurie; A.C.Allen, J. Chromatogr., 292 (1984), 283.
42) R.D.Dandedau and E.M. Zenner, J. High Res. Chromatography, 2 (1979),
351.
43) M.Gloger and H.Neurnann, Forensic Sci. Int., 22 (1983), 63.
44) LS.Lurie and S.M.Carr, J. Liq. Chrornatogr.,6 (1983), 1617.
45) 1.S.Lurie and S.M.Carr, J. Liq. Chromatogr., 9 (1986), 2485.
46) I.S.Lurie, A.C.Allen, J. Chromatogr., 3 17 (1 984), 427.
47) LS.Lurie, D.A.Cooper and R.F.Klein, J. Chromatogr., 598 (1992), 59.
48) I.S.Lurie, A.RSperling and R.P.Meyers, J. Forensic Sci., 39 (1994), 74.
49) J.K.Baker, R.E.Skelton and C.Y.Ma, J. Chromatogr., 168 (1979), 417.
50) P.T.Kissinger, Electroanalysis, 4 (1992), 359.
51) M.J.Milano, S.Lam and E.Grushka, J. Chromatogr., 125 (1976), 315.
52) L.Yang, G.L.Fergusson and M.L.Vestal, Anal. Chem., 56 (1984), 2632.
53) D.M.Demorest, J.C.Fetzer, I.S.Lurie, S.M.Carr, K.B.Chatson, LCGC, 5
(1987), 128.
54) C.M. Selavka, I.S.Krul1 and I.S.Lurie, Forensic Sci. Int., 31 (1986), 103.
55) I.S.Lurie, D.A.Cooper and I.S.Krull, J. Chromatogr., 629 (I 993), 143.
56) P.A.Peaden, J.C.Fjeldsted, M.L.Lee, S.R.Springston and M.Novotny,
Anal. Chem., 54(1982), 1090.
57) T.A.Berger, Packed Column SFC, 'he Royal Society of Chemistry,
Cambridge, 1995.
58) I.S. Lurie, LCGC, 6 (1988), 1066.