Ultra Performance Liquid Chromatography (UPLC) improves

chromatographic resolution, speed and sensitivity analysis. It uses fine particles

and saves time and reduces solvent consumption 11-51. The UPLC comes from

HPLC. The HPLC has been the evolution of the packing materials used to effect

the separation. An underlying principle of HPLC dictates that as column packing

particle size decreases, efficiency and thus resolution also increases. As particle

size decreases to less than 2 . 5 p , there is a significant gain in efficiency and it

doesn't diminish at increased linear velocities or flow rates according to the

common Van Deemter equation [6]. By using smaller particles, speed and peak

capacity (number of peaks resolved per unit time) can be extended to new limits

which is known as Ultra Performance.

The classic separation method of HPLC (High Performance Liquid

Chromatography) has many advantages like robustness, ease of use, good

selectivity and adjustable sensitivity. It's main limitation is the lack of efficiency

compared to gas chromatography or the capillary electrophoresis [7, 81 owing to

low diffusion coefficients in liquid phase, involving slow diffusion of analytes in

the stationary phase. The Van Deernter equation shows that efficiency increases

with the use of smaller size particles but this leads to a rapid increase in back

pressure, while most of the HPLC systems can operate only up to 400 bars. That is

why short columns filled with particles of about 2 p are used with these systems

to accelerate the analysis without loss of efficiency while maintaining an

acceptable loss of load.

To improve the efficiency of HPLC separations, the following can be

done:-

A. work at higher temperatures

B, use of monolithic columns

5.2 APPLICATIONS OF UPLC

5.2.1 Principle

The UPLC is based on the principal of use of stationary phase consisting

of particles less than 2 pn (while HPLC columns are typically filled with particles

of 3 to 5 pn). The underlying principles of this evolution are governed by the Van

Deemter equation, which is an empirical formula that describes the relationship

between linear velocity (flow rate) and plate height (HETP or column efficiency)

[9]. The Van Deemter curve, governed by an equation with three components,

shows that the usable flow range for a good efficiency with a small diameter

particles is much greater than for larger diameters [I 0-121.

H=A+B/v+Cv

where A, B and C are constants and v is the linear velocity, the carrier gas flow

rate. The A term is independent of velocity and represents "eddy" mixing. It is

smallest when the packed column particles are small and uniform. The B term

represents axial diffusion or the natural diffusion tendency of molecules. This

effect is diminished at high flow rates and so this term is divided by v. The C term

is due to kinetic resistance to equilibrium in the separation process. The kinetic

resistance is the time lag involved in moving from the gas phase to the packing

stationary phase and back again. The greater the flow of gas, the more a molecule

on the packing tends to lag behind molecules in the mobile phase. Thus this term

is proportiond to v.

Therefore it is possible to increase throughput and thus the speed of

analysis without affecting the chromatographic performance. The advent of UPLC

has demanded the development of a new instrumental system for liquid

chromatography, whi'ch can take advantage of the separation performance (by

reducing dead volumes) and consistent with the pressures (about 8000 to 15,000

PSI, compared with 2500 to 5000 PSI in HPLC). Efficiency is proportional to

column length and inversely proportional to the particle size [13]. Therefore, the

column can be shortened by the same factor as the particle size without loss of

resolution. The application of UPLC resulted in the daection of additional drug

metabolites, superior separation and improved spectral quality 114,151.

533 Charactcristica comparison between UPLC and HPLC

The characteristics comparison between UPLC and HPLC arc presented in

the Table 5.1.

Table 5.1

Comparison between UPLC and HPLC

5.23 Sample injection

Comparison between UPLC and HPLC

In UPLC, sample introduction is critical. Conventional injection valves,

either automated or manual, are not designed and hardened to work at exrreme

pressure. To protect the column from extreme pressure fluctuations, the injection

process must be relatively pulse-free and the swept volume of the device also

needs to be minimal to reduce potential band spreading. A fast injection cycle

time is needed to fully capitalise on the speed afforded by UPLC, which in turn

requires a high sample capacity. Low volume injections with minimal carry over

are also required to increase sensitivity [16]. There are also direct injection

approaches for biological samples [17,18].

Characteristics Particle size Maximum backpressure Analytical column

Column dimensions Column temperature Injection volume

5.2.4 UPLC columns

Figure 5.1 presents UPLC columns. Resolution is increased in a 1.7 pn

particle packed column because efficiency is better. Separation of the components

of a armplc requires a bonded phase that provides both retention i d selectivity.

Four bonded p h u n are available for UPLC qwntions: ACQUITY UPLCTU

BEH C18 and C8 (straight chain alLy1 columns), ACQUITY UPLC BEH Shield

RPl8 (embedded polar group column) and ACQUITY UPLC BEH Phenyl

(phenyl group tethed to the silyl functionality with a C6 allryl) [19]. Each

HPLC 3 to 5m 35-40 MPa Alltima C,,

150 X 3.2 mm 30 "C S@(Std.inlOO% MeOH)

UPLC Less than 2m 103.5 MPa Acquity UPLC BEH C18

50 X 2.1 mm 65 "C 2pL (Std.InIOO% Me0

column chemistry provides a diffmt combination of hydrophobicity, silanol

activity, hydrolytic stabiity and chemical intention with analytes. ACQUITY

UPLC BEH C18 and C8 columns are considered the universal columns of choice

for most UPLC separations by providing the widest pH range. They incorporate

trifunctional ligand bonding chemistries which produce superior low pH stability.

This low pH stabiity is combined with the high pH stability of the 1.7 pn BEH

particle to deliver the widest usable pH operating range. ACQUITY UPLC BEH

Shield RPlS columns ate designed to provide selectivities which complement the

ACQUITY UPLC BEH C18 and C8 phases. ACQUITY UPLC BEH Phenyl

columns utilize a tribctional C6 alkyl tether between the phenyl ring and the

silyl functionality. This ligand, combined with the same proprietary end capping

processes as the ACQUITY UPLC BEH C18 and C8 columns, provides long

column lifetimes and excellent peak shape. This unique combination of ligand and

end capping on the 1.7 pn BEH particle creates a new dimension in selectivity

allowing a quick match to the existing HPLC column. An internal dimension (ID)

of 2.1 mm column is used. For maximum resolution, choose a 100 mm length and

for faster analysis, and higher sample throughput, choose 50 rnm column.

Qis -AOQWTY UPLCn BEH C18

ACQUITY UPLCw BEH C8

ACQUJTY UPLCw BEH Shldd RP18 k.AAAwv

Fig. 5.1 A C Q W UPLC beh Column ehmistrlea

Half-height peak widths of less than one second are obtained with 1 . 7 ~

particles, which give significant challenges for the detector. In order to integrate

an analyte peak accurately and reproducibly, the detector sampling rate must be

high enough to capture enough data points across the peak. The detector cell must

have minimal dispersion (volume) to preserve separation efficiency. Conceptually,

the sensitivity hawse for UPLC detection should be 2-3 times higher than HPLC

separations, depending on the detection technique. The MS detection is

significantly enhanced by UPLC, increased peak concentrations with reduced

chromatographic dispersion at lower flow rates promotes increased source

ionization efficiencies.

The ACQUITY UPLC System consists of a binary solvent manager,

sample manager including the column heater, detector, and optional sample

organiser. The binary solvent manager uses two individual serial flow pumps to

deliver a parallel binary gradient. There are built-in solvent select valves to choose

from up to four solvents. There is a 15,000-psi pressure limit (about 1000 bar) to

take full advantage of the sub-2pn particles. The sample manager also

incorporates several technology advancements. Using pressure assisted sample

introduction, low dispersion is maintained through the injection process, and a

series of pressures transducers facilitate self-monitoring and diagnostics. It uses

needle-in-needle sampling for improved ruggedness and needle calibration sensor

increases accuracy. Injection cycle time is 25 seconds without a wash and 60 see

with a dual wash used to further decrease carry over. A variety of microtiter plate

formats (deep well, mid height, or vials) can also be accommodated in a

thermostatically controlled environment. Using the optional sample organiser, the

sample manager can inject from up to 22 microtiter plates. The sample manager

also controls the column heater. Column temperatures up to 65OC can be attained.

To minimise sample dispersion, a "pivot out" design allows the column outlet to

be placed in closer proximity to the source inlet of an MS detector [20].

For UPLC detection, the tunable WNisible detector is used which

includes new electronics and firmware to support Ethernet communications at the

high data rates. Conventional absorbance-based optical detectors are concentration

sensitive detectors, and for UPLC use, the flow cell volume would have to be

reduced in standard WNisible detectors to maintain concentration and signal.

According to Beer's Law, srnaller volume conventional flow cells would also

reduce the path length upon which the signal strength depends. A duction in

cross-section means the light path is reduced and transmission drops with

increasing noise. Therefore, if a conventional HPLC flow cell were used, UPLC

sensitivity would be compmmised. The ACQUITY Tunable UVNisible detector

cell cpnsists of a light guided flow cell equivalent to an optical fiber. Light is

efficiently transferred down the flow cell in an internal reflectance mode that still

maintains a lOmm flow cell path length with a volume of only 500 mL. Tubing

and connections in the system are efficiently routed to maintain low dispersion

and to take advantage of leak detectors that interact with the software to alert the

user to potential problems [21].

5.2.5 Advantages

The major advantages of the UPLC are

It decreases run time and increases sensitivity

It provides the selectivity, sensitivity, and dynamic range of LC analysis

It maintaining resolution performance.

It expands scope of Multiresidue Methods

UPLC's fast resolving power quickly quantifies related and unrelated

compounds

Faster analysis through the use of a novel separation material of very fine

particle size

Operation cost is reduced

Less solvent consumption

Reduces process cycle times, so that more product can be produced with

existing resources

Increases sample throughput and enables manufacturers to produce more

material that consistently meet or exceeds the product specifications,

potentially eliminahg variability, failed batches, or the need to re-work

material [22,23]

Delivers real-time analysis in step with maaufscturing processes

Assures end-product quality, includii fiaal release testing

5.2.6 Diiadvantages

Due to increased pressure requires more maintenance and reduces the life

of the wlumns of this type. So far performance similar or even higher has been

demonstrated by using stationary phases of size around 2 pn without the adverse

effects of high pressure. In addition, the phases of less than 2 pn are generally

non-regenerable and thus have limited use [24,25].

5.2.7 Analysis of natural products and traditional herbal medicine

The UPLC is widely used for analysis of natural products and herbal

medicines. For traditional herbal medicines, also known as natural products or

traditional Chinese medicines, analytical laboratories need to expand their

understanding of their pharmacology to provide evidence-based validation of their

effectiveness as medicines and to establish safety parameters for their production.

The main purpose of this is to analyze drug samples arising from the complexity

of the matrix and variability from sample to sample. Purification and qualitative

and quantitative chromatography and mass spectrometry are being applied to

determine active drug candidates and to characterize the efficacy of their

candidate remedies. The UPLC provides high-quality separations and detection

capabilities to identify active compounds in highly complex samples that results

fiom natural products and traditional herbal medicines. Metabonomics-based

analysis, using UPLC, exact mass MS and Marker Lynx Soflware data processing

for multivariate statistical analysis, can help quickly and accurately characterize

these medicines and also their effect on human metabolism. Preparative-scale

fractionation and purification is used along with classic quantitative bioanalytical

tools used in drug development.

5.2.8 IdenM~cation of Metabolite

Biotransformation of new chemical entities WE) is necessary for drug

discovery. When a compound reaches the development stage, metabolite

identification becomes a ngulated process. It is of utmost importance for lab to

sumssfidly detect and identif) all circulating metabolites of a cadi i drug.

Discovery studies are generally amied out in vim to identify Nor metabolites

so that metabolic weak spots on the drug candidate molecule can be ncognized

and protected by changing the compound structure. Key for analysts in metabolite

151

identification is maintaining high sample throughput and providing results to

medicinal chemists as soon as they are available. UPJ.,C/MS/MS addresses the

complex analytical requirements of biomarker discovery by offering unmatched

sensitivity, resolution, dynamic range and mass accuracy.

53.9 Study of metabonomics I metabolomica

Metabonomics studies are carried out in labs to accelerate the development

of new medicines. The ability to compare and contrast large sample groups

provides insight into the biochemical changes that occur when a biological system

is exposed to a new chemical entity (NCE). Metabonomics provides a rapid and

robust method for detecting these changes improves understanding of potential

toxicity and allows monitoring the efficacy. The correct implementation of

metabonomic and metabolomic information helps similar discovery, development

and manufacturing processes in the biotechnology and chemical industry

companies. With these studies, scientists are better able to visualize and identify

fundamental differences in sample sets. The UPLC/MS System combines the

benefits of UPLC analyses, high resolution exact mass MS, and specialized

application managers to rapidly generate and interpret information-rich data,

allowing rapid and informed decisions to be made.

5.2.10 ADME (Absorption, Distribution, Metabolism and Exereation)

Screening

Phannacokinetics studies include studies of ADME (Absorption,

Distribution, Metabolism and Excreation). ADME studies measure physical and

biochemical properties - absorption, distribution, metabolism, elimination, and

toxicity of drugs where such compounds exhibit activity against the target disease.

A significant number of candidate medicines fall out of the development process

due to toxicity, If toxic reactions or any side effect occurs in the

discoveryldevelopment process, then it becomes more costly. It is difficult to

evaluate candidate drugs for possible toxicity, drugdrug interactions, inhibition

andlor induction of metabolizing mymes in the body. Fail~lre to properly identify

these potential toxic events can cause a compound to be withdrawn from the

market. The high resolution of UPLC enables accuntte detsction and integration of

peaks in complex matrices and extra sensitivity allows peak detection for samples

generated by lower concentration incubations and sample pooling. These an

important for automated generic methods as they reduce failed sample analyses

and save time.

UPLC/MS/MS provides following hdvantages.

UPLC can more than double throughput with no loss in method robustness.

UPLC is also simpler and more robust than the staggered separations sometimes

applied with HPLC methods.

Tandem quadruple MS provides sensitivity and selectivity for samples in

matrix using multiple reaction monitoring (MRM) for detection and automated

compound optimization. The UPLC/MS/MS operation with rapid and generic

gradients has been shown to increase analytical throughput and sensitivity in high

throughput phmacokinetics or bioanalysis studies, including the rapid

measurement of potential p450 inhibition, induction and drug-drug interactions.

As well, since this UPLC-based approach can help labs pre-emptively determine

candidate toxicity and drug-drug interactions, it enables organizations to be more

confident in the viability of candidate medicines that do progress to late-stage

clinical trials. Tandem quadruple MS combines with UPLC in ADME screening

for sensitivity and selectivity with fast analyses of samples in matrix to be

achieved with minimal cleanup, using MRM (multiple reaction monitoring) for

detection and automated compound optimization.

5.2.11 Bioanalysis 1 Bioequivalence studies

For pharmacokinetic, toxicity and bioequivalence studies, quantitation of a

drug in biological samples is an important part of development programs. The

drugs are generally of low molecular weight and are tested during both preclinical

and clinical studies. Several biological matrices are used for quantitative

bioanalysis, the most common being blood, plasma and urine [26]. The primary

technique for quantitative bioanalysis is LC/MS/MS. The sensitivity and

selectivity of UPLCMSMS at low detection levels generates wmte and

reliable data that can be used for a variety of differmt purposes, including

statistical phannacokinetics (PK) analysis. Developing a robust and compliant

LC/MS/MS assay has traditionally been the domain of very e x p e r i d analysts.

UPLC/MS/MS kips in the processes of method development for bioanalysis into

153

logical steps for MS, LC and sample preparation. QuantiQtive bioanalysis is also

an integral part of bioequivalence studies, which are used to determine if new

formulations of existing drugs allow the compound to reach the bloodstream at a

similar rate and exposure level as the original formulation. UPLCMSMS

solutions are proven to increase efficiency, productivity and profitability for

bioequivalence laboratories. Applications of UPLC/MS/MS in bioequivalence and

bioa@ysis are:-

> In UPLCIMSIMS, LC and MS instruments and software combine in a

sophisticated and integrated system for bioanalysis and bioequivalence

studies, providing an unprecedented performance and compliance support.

P UPLC/MS/MS delivers excellent chromatographic resolution and

sensitivity.

> MS delivers simultaneous full-scan MS and multiple reaction monitoring

(MRM) MS data with high sensitivity to address matrix monitoring.

> UPLC Sample Organizer maximizes efficiency by accommodating large

numbers of samples in a temperature-controlled environment, ensuring

maximum throughput.

> Increase the sensitivity of analyses, quality of data including lower limits

of quantitation (LLOQ), and productivity of laboratory by coupling the

UPLC System's efficient separations with fast acquisition rates of tandem

quadruple MS systems.

R Easily acquire, quantify and report full system data in a compliant

environment using a security-based data collection software

P Ensure the highest quality results and reliable system operation in

regulated environment

5.2.12 Diwolution tssting

For quality cantml and release in drug wufacturing, didissolution testing

is essential in the formulation, development and production process. In sustained-

release dosage formulations, testing higher potency drugs is perticularly important

where dissolution can be the rate-limiting step in medicine delivery. The

dissolution profile is used to demonstrate reliability and batch-to-batch uniformity

of the active ingredient. Additionally, newer and more potent formulations require

increased analytical sensitivity. The UPLC provides precise and reliable

automated online sample acquisition. It automates dissolution testing, from pill

drop to test start, through data acquisition and analysis of sample aliquots, to the

management of test result publication and distribution.

5.2.13 Foreed degradation studies

One of the most important factors that impacts the quality and safety of

pharmaceuticals is chemical stability. The FDA and ICW require stability testing

data to understand how the quality of an API (active pharmaceutical ingredient) or

a drug product changes with time under the influence of environmental factors

such as heat, light, pressure and moisture or humidity. Knowledge of these

stability characteristics defines storage conditions and shelf - life, the selection of

proper formulations and protective packaging, and is required for regulatory

documentation. Forced degradation or stress testing, is carried out under even

harsher conditions than those used for accelerated stability testing. Generally

performed early in the drug development process, laboratories cause the potential

drug to degrade under a variety of conditions like peroxide oxidation, acid and

base hydrolysis, photo stability and temperature to understand resulting

byproducts and pathways that are necessary to develop stability indicating

methods. The most common analytical technique for monitoring f o r d

degradation experiments is HPLC with UV andlor MS detection for peak purity,

mass balance and identification of degradation products but these HPLC-based

methodologies are t i m m u m i n g and provide only medium resolution to ensure

that all of the degradation products are accurately detectad, The PDAIMS

(photodiode array and MS) allows for faster and higher peak capacity q a d o a s

and for complex degradation product profiles too. C o m b i i the

chromatographic speed, resolution, and sensitivity of UPLC scpadoas with the

high-speed scan rates of UPLC-spccific photodiode array and MS detection will

give confidence for identifying degradation products and thus shorkning the time

required for developing stability-indicating methods.

5.2.14 Manufacturing 1 QA 1 QC

Identity, purity, quality, safety and efficacy are the important factors to be

considered while manufacturing a drug product. The successful production of

qualit); pharmaceutical products requires raw materials which meet purity

specifications. Those final pharmaceutical products meet, and hopefully exceed,

defined release specifications. Continued monitoring of material stability is also a

component of quality assurance and control. The UPLC is used for the highly

regulated, quantitative analyses performed in QA/QC laboratories. The supply of

consistent, high quality consumable products plays an important role in a

registered analytical method. The need for consistency over the lifetime of a drug

product which could be in excess of 30 years is essential in order to avoid method

revalidation and associated production delays.

5.2.15 Method development 1 validation

According to FDA, validation is defined as an establishing documented

evidence that provides a high degree of assurance that a specific process will

consistently produce a product meeting its predetermined specifications and

quality attributes. Method development and validation is a time-consuming and

complicated process: labs need to evaluate multiple combinations of mobile

phase, pH, temperature, column chemistries, and gradient profiles to arrive at a

robust, reliable separation for every activity.

The UPLC help in critical laboratory function by inneasing efficiency,

reducing costs and improving opportunities for business success, The UPLC

column chemistries can easily translate across analytical- and preparativ~scale

separation tasks. The UPLC provides efficiencies in method development. Using

UPLC, aualysis times becomes as short as one minute, methods can be optimized

in just one or two bouts, significantly reducing the time rcquirad to devclop and

validate with UPLC, sepadon speed and efficiency allows for the rapid

development of methodologies.

The following parts of UPLC an important to give the rcquired information:-

> UPLC columns: High allows for a wide range of column

tempemtms and pHs to be e x p l d .

> UPLC Column Manager: Easily evaluate column temperatures from 10 OC

below room temperature to 90 'C, enables to use HPLC methods on the

, UPLC before scaling to UPLC.

P UPLC Calculator: Put information at fingertips about how to transition

existing chromatographic analyses to faster UPLC methods.

5.3 PROSTAGLANDINS

The name prostaglandin derives h m the prostate gland. When

prostaglandin was first isolated from seminal fluid in 1935 by the

Swedish physiologist Ulf Von Euler, [27] and independently by M.W. Goldblatt

[28], it was believed to be part of the prostatic secretions (In fact, prostaglandins

are produced by the seminal vesicles). It was later shown that many other tissues

secrete prostaglandii for various functions. The first total synthesesof

prostaglandin Fz, and prostaglandin Ez were reported by E. J. Corey in 1969 [29].

Prostaglandins are unsaturated carboxylic acids, consisting of a 20 carbon

skeleton that also contains a five member ring and are based upon the fatty acid,

arachidonic acid. There are a variety of structures one, two or three double bonds.

On the five member ring there may also be double bonds, a ketone or alcohol

groups. The prostaglandins are a family of lipid-soluble, unsaturated hydroxy

acids containing twenty carbon (C) atoms and based on the prostanoic acid

skeleton. The C atoms in the molecule are numbered as shown in Fig. 5.2.

The pmstaglandins are grouped according to the chemical groups on the

pentane ring into types designated by the letters E, F, A, B, C and D. E-type

prostaglandins have a ketone p u p at C9 and a hydroxyl p u p at Cli. In D-type

prostaglandins theae groups are reversed, the ketone being at C1I and the hydroxyl

at C9. F-type prostaglandins have hydroxyl groups at C9 and Cli and fonn isomers

designated x and 3, according to the plane of the hydroxyl groups. The A, B and

C-type prostaglandins have a ketone group at C9 and 10-1 1, 11-12 and 8-12

double bonds respectively. The prostaglandins are also grouped into mono-, bis-

01- tris-unsaturated classes according to the number of carbonarbon double bonds

in the side chain. The mono-unsaturated prostaglandins have only a 13- 14 double

bond, the bis-unsaturated have a 5-6 double bond in addition, and the

trisunsaturated have a 17-18 double bond also [30] (Andersen, 1971).

Prostaglandins E, F, A, B, C, D have a carboxyl group at C1 and a secondary

alcohol group at C15. Prostaglandins are not stored in tissues in a preformed state

but are synthetized and released in response to a given stimulus[3 I ] (Piper and

Vane, 1971). All types of mammalian tissue that have been examined are capable

of synthetizing at least a small quantity of prostaglandins. Prostaglandins are

synthetized from eicosatrienoic (dihomo-y-linolenic), eicosatetraenoic

(arachidonic) or eicosapentanoic acids.

These compounds are incorporated in phospholipids and are converted to

prostaglandins by the action of cyclo-oxygenase [32](Samuelsson, 1976). This

enzyme, previously known as prostaglandin synthetase, is present in the

microsoma1 fraction of cells and initially brings about cycliuttion and inclusion of

molecular oxygen in the precursor. The cyclooxygenase initially stimulates

formation of the cyclic-endoperoxides, prostaglandins G2 and H2. Prostaglandins

G2 and H2 are then converted to either prostaglandins E2, D2 and F2. Or

thromboxane A2 which is unstable and decays to thromboxane B2; in some

tissues, e.g. lung or platelets, the main products of arachidonic acid metabolism

are thromboxanes rather than prostaglandins [33] (Hambexg, Svensson and

Samuelsson, 1976). Conversion of the eadopetoxides to thromboxanes is

enymically controllad by thromboxane synthetase (Needlunaa, 1976). As

recently shown by Moncada (1976), when prostagldh 02 or HZ act on blood

vessel walls prostscyclin @m@hdh 12) is formed cmymtically. The stab16

metabolite of prostacyclin is 6-oxopwstaglaadin Flj. The posib'ity exists for

some inter coaversion of prostaglandins, for example prostaglandin E2 may be

reduced to prostaglandin M, by the action of 94x0-rcductase which is present in

tissues of a number of species [34] (Hensby, 1974). E-type prostaglandins are

converted to A-type by acid or alkaline conditions (outside the range pH 5-8) and

A-type rearranges to B in alkaline solution (Andersen, 1971). An isomerase

present in plasma of some species converts prostaglandins A first to prostaglandin

C and then to B [35] (Jones, 1972). Prostaglandins of the E and F series have a

short half-life in the circulation and lose up to 95% of their biological activity on

one passage through the pulmonary circulation and are further degraded on

passage through other vascular beds [36](Ferreira and Vane, 1967). Initially the

prostaglandins are taken up fiom the vascular space [37] (Bito, 1975) and then

metabolized by a series of enzymes to metabolites which usually have less

biological activity than the prostaglandins [38] (Crutchley and Piper, 1975), 15-

OH prostaglandin dehydrogenase causes the oxidation of the secondary alcohol

group at C15 which is the &-limiting step in the breakdown of prostaglandins.

A-type prostaglandins are not so rapidly metabolized by prostaglandin

dehydrogenase and do not lose activity in the pulmonary circulation but in the

hepatic portal circulation [39] (Horton and Jones, 1969). A-type prostaglandins

lose activity on conversion to prostaglandin B which is biologically inactive. The

pulmonary metabolites of the prostaglandins are further broken down in the liver

and kidney by n-oxidation to dinor (eighteen C atoms) and tetranor (sixteen C

atoms) derivatives and finally undergo 5-oxidation (Samuelsson, 1971). The

resulting metabolites are excreted in the urine and are biologically inactive.

Prostagfandins ( P a ) have been identified as a heterogenous group of

hormones which show extremely diverse biological activities. First descriptions

reporting the activity of prostaglandins appeared in the early 1930s by Kumok

and Lieb 1401 and Goldblatt [dl]. Twenty years later the firat prostaglandins, PGE

and PGF, were purified [42]. In most animals, arachidonic acid is the most

important precursor of PGs. Beside the original two POs, a wly of PGa could be

ideatifled named alphabetically h m PGA to PGH. Receptors and receptor

aflinity on the basis of functional data have bem f o d out for the PGa D , E , F , I by Kermody 1982 [43] and Coleman 1984 [44]. Further ligand binding studies

were performed in the 1970s and 1980s. Second messenger studies were initially

focused on cyclic adenosinemonophosphate (CAMP) for the E-series PGs [45].

One example for a CAMP mediated action by PGE is the effect on platelets which

is interestingly distinct: PGE caused an elevation of platelet CAMP, whereas PGE

caused a reduction That means that both, inhibitory and stirnulatory effects on

platelet aggregation are stimulated by two distinct E-series PGs, PGE and PGE

respectively. Whereas the effects of PGE and PGE relied on CAMP were denied

for PGF [46]. It has been accepted that cyclic guanosine 3', 5'-monophosphate

mediated the action of PGF, a concept which has been questioned recently. PGF

has been characterized as a potent FP receptor agonist, although not very

selective. Binding properties to channel EP and TP receptors have also been

reported. Fluprostenol and cloprostenol are selective PGF analoges, synthetized in

the 1970s [47]. The existence of FP receptors has been demonstrated to exist in a

variety of distinguished tissues over a wide range of different species. One tissue

in which the FP receptors seems to present throughout all species, even in the

human being, is the corpus luteurn. In some rodents, excluding guinea pig and in

human beings contractile FP receptors of the myometrium are reported [48]. In

dogs and cats, FP receptors of airway smooth muscles cells are described with

contractile properties mediated [49]. Similar effects of contraction are known for

the mesangial cells of the kidney glomerulus [50] and coronary arteries [51]. In

perhsed rat hearts, increase in global contractility has been studied.

5.3.1 Classification of prostaglandins

The classification of prostaglandins (figure 5.5) as local or cellular,

hormones is justified by their varied functions and the absence of a special organ

for their biosynthesis. Their mechanism of action is still unclear. It has been

established that prostaglandins affect the activity of the enzyme adenyl cyclase,

which regulates the concentration of cyclic adenosine 3', 5'-monophosphate

(cyclic AMP) in the cell. Since prostagladins influeace the biosyntbcsia of cyclic

AMP and cyclic AMP participates in hormonal regulation, a possible mechanism

of action of prostaglandins could consist in comcting (intensifjing or weakening)

the action of other hormones.

Pmstanoic acid

Fig.53 The classification of prostaglandins

5.3.2 Receptors for prostaglandin E2

Prostaglandin E2 (PGEz) is generated by the sequential metabolism of

arachidonic acid by cyclo-oxygewe and prostaglandin E synthase [52, 531. This

lipid mediator has pleiotropic actions in a range of tissues, including the immune

system [54]. Within the immune system, PGE2 modulates the functions of cell

populations, such as T cells and macrophages, which are critical to the immune

response. For example, PGE2 suppresses proliferation of human T cells [55, 561.

In rnacrophages, PGE2 inhibits production of cytokines such as TNF-a and IL-12

[57,58] and alters antigen presentation by inhibiting expression of MHC class I1

prateins [59]. Thus, the overall actions of PGE2 on in vitro models of cellular

immune responses tend to be inhibitory and suppressive [60]. Along with its

actions to inhibit cellular Mans, P G b may also affect the overall character of

an immune response. PGbmay polarize cellular response tow a Th2

phenotype enhancing IL-4 and IL5 production [61] and facilitating

imrnuuoglobulin class switching to IgE [62].

These actions of PG& can dramatically alter the outcome of immune

responses in the intact organism. For instance, PGE2 has inhibitory and protective

effects in autoimmune disease. In murine lupus models, administration of

PGEz and its analogues improves survival [63,64]. This improvement in survival

is accompanied by reduced auto-Ab production and a substantial reduction in

immune-mediated kidney injury. Similarly, PGE2 may delay or prevent allograft

rejection. In a rat model of kidney transplantation, admistration of

PGEl markedly prolonged graft survival and reduced systemic cellular

alloirnmune responses [65]. Analogous effects of PGE2 to ameliorate rejection

have been observed in animal models of heart, intestinal and skin transplantation

[66, 671. In human renal transplant recipients, a reduced number of kidney

allograft rejection episodes have also been reported with PGEl analogues [68].

5 3 3 Functions of Prostaglandins

There are a variety of physiological effects including

1. Activation of the inflammatory response, production of pain, and fever.

When tissues are damaged, white blood cells flood to the site to try to

minimize tissue destruction. Prostaglandins are produced as a result.

2. Blood clots form when a blood vessel is damaged. A type of prostaglandin

called thromboxane stimulates constriction and clotting of platelets.

Conversely, PGU is produced to have the opposite effect on the walls of

blood vessels where clots should not be forming.

3. Certain prostaglandins are involved with the induction of labour and other

reproductive processes. PGE2 causes uterine contractions and has been

used to induce labour.

4. Prostaglandins are involved in several other organs such as the

gastrointestinal tract (inhibit acid synthesis and increase sexdon of

protective mucus), increase blood flow in kidneys and lcukoeims promote

constriction of bronchi associated with asthma

5.4 BJMATOPROST AND THEIR IMPURITIES

Bimatoprost (7-[3,5-dihydroxy-2- (3-hydroxy-5-phenyl-pent-1enyl)-

cyclopentyl]-N-ethyl-hept-5-enamide), (sold in the U.S., Canada and Europe

by Allergan, under the trade name Lumigan) is a promglandin analog/prodrug

used topically (as eye drops) to control the progression ofglaucoma and in the

management of ocular hypertension. It reduces intraocular pressure (i0P) by

increasing the outflow of aqueous fluid h m the eyes [69]. It has also been used

and prescribed off-label to lengthen eyelashes [70]. In December 2008, this use

was approved by the U.S. Food and Drug Administration; the cosmetic

formulation of bimatoprost is sold as Latisse [71] recently, at least three case

series have suggested that bimatoprost has the ability to reduce adipose (fat)

tissue. Its molecular formula is C25H31N04 and its chemical structure as below and

developed for treatment of glaucoma [72, 731. The name prostaglandin dcrives

from the prostate gland. When prostaglandin was first isolated from seminal

fluid in 1935 by the Swedish physiologist Ulf von Euler [74] and independently

by M.W. Goldblatt [75], it was believed to be part of the prostatic secretions. (In

fact, prostaglandins are produced by the seminal vesicles). It was later shown that

many other tissues secrete prostaglandins for various functions. The first total

syntheses of prostaglandin F2a and prostaglandin E2 were reported by E. J.

Corey in 1969 [76]. It reduces intraocular pressure by increasing uveoscleral

outflow of aqueous humour as a result of stimulating prostanoid selective FP

receptors [77, 781. Bimatoprost and Bimatoprost is administered in therapy of

primary open - angle glaucoma and ocular hypertension [79] and its efficacy has

been evaluated on human and animal healthy and glaucomatous eyes [80 - 831.

Impurities related to the organic synthesis which may be present in

Bimatoprost bulk drug substance include 15(R) Bimatoprost (impurity-I; 7-133-

dihydroxy-2- (15 R - tri hydroxy-5-phenyl-pent-1-enyl) cyclopentyl]-N-ethyl-

hept-5-enamide),) Birnatoprost acid (Impurity-11; (52)-7-((lR,2%3R,5S>3,5di

hydroxy-2-((S$)3-hydroxy-5-phenylpeat-I a y l ) cyclopentyl) hept-5-enoic acid)

Bimatoprost Kw~llpurity-III), Bimatoprost methyl (Impurity - N(5Z) methyl-

7-((1~2~3~5S)3,5-dihydroxy-2-((S$~3-hydroxy-5-phylpt-l-

enyl)cyclopentyl) hept-5-enoate) and Bima!oprost with the double bond between

carbon atom 5 and 6 changed h m cis (2) to trans (E). The chemical fwmulae of

these impurities are also presented Fig. 5.4.

Bimatoprost

Bimatoprost Acid

15- R Bimatoprost

Bimatoprost Keto

Methyl ester impurity

Fig5.4 Structum of Bimatoprost, lsomcra and impurities

It is necessary to develop a selective method for analysis of all the known

isomers of bimatoprost that could arise during synthesis and w e .

Undoubtedly, the HPLC coupled with spectroscopic methods, is one of the most

h q d y used methods for sepmtion, quantification, and identification of drug impurities. Several HPLC procedures for analysis of p r o s t a w analogs have

been nportcd. Wborton and co-workers found that whams rev&-phase

chromatography separated PGEl and PGE2, which differed by the pmence of an

additional double bond in the alkyl chain of E2, it did not separate the geometrical

isomers PGE2 and PGD2, which were separated on the silica column [84, 851.

Several other, related, pros taglandins have been achieved the reversed-phase

separation of cloprostenol (an analog prostaglandin PGF2a) from the

stereoisomers 15epi-cloprostenol trans-cloprostenol. Very few HPLC procedures

for analysis of Bimatoprost & Birnatoprost have been reported. Ashfaq recently

reported a simple and rapid RP-18 method quantification of Bimatoprost and its

metabolite (the fiee acid of Bimatoprost) in pharmaceutical formulations, whereas

Morgan have reported HPLC method for simultaneous determination of

Bimatoprost and acid during examination of the effects of controlled heat and UV

exposure on the stability of Bimatoprost. None of these papers mentioned the

impurities I, 11, I11 and IV. To the best our knowledge, there are no published

reports of analysis of Bimatoprost in the presence of impurities I, 11, III and IV.

The objective of the work discussed in this paper is to establish validated UPLC

method for analysis of the isomers of Bimatoprost.

5.5 AIMS AND OBJECTIVES

The present chapter has proposed the following objects for Development

and Optimization of a Novel Method Developed Bimatoprost by Ultra

Performance Liquid Chromatography (UPLC)

1. Bimatoprost, impurities and isomer separation by the UPLC.

2. An improved LC-MSIMS method for quantitative determination of

Birnatoprost methyl ester

3. Method validation of Bimatoprost by the UPLC method,

5.6 EXPERIMENTAL

5.6.1 Materials and Reagenta

The samples of Bhatopmst bulk material investigated wm obtained from

the F,nnanthi labs (Hyderabad). Reference standards of Bimatoprost and impurities

I, 11, III aud IV were obtained h m Ennanthi labs (Hyderabad). The solvents (of

n-Hexane, dehydrated alcohol and methanol) wan of HPLC grade Jtandard.

5.6.2 Chromatographic Conditions

By UPLC on Aquity UPLC BEH Shield RP 18 (100 X 2.1 mm), 1.7pm

and detector of UV at 237m, with a homogeneous mixture of n-Hexane,

dehydrated alcohol and methanol 80: 10: 10 (vfv), before use the mobile phase was

filtered through a 0.45-pn filter and degassed under vacuum. The flow rate was 1

mL min-1 and the injection volume was 20 &. Solutions of Bimatoprost and

impurities I, 11,111 and IV were prepared by dissolving appropriate amounts of the

bulk substance in the mobile phase. The retention times of the compounds were

examined by injection of standard solutions.

5.7 RESULTS AND DISCUSSION

5.7.1 LC-Mass Condition

The API4000 triple quadruple mass spectrometer was operated under the

positive electrospray ionization mode (ESI'). The mass spectrometer was tuned by

infusion of Bimatoprost impurity (methyl ester) (1.0 U m L ) in the mobile phase

at a flow rate of 10 f lmin with a syringe pump (Harvard Apparatus, South

Natick, MA, USA). The tandem mass spectrometer was tuned to monitor

the mlz -( 402, Mt1+ 403 and MtNa m/z-+ 184.0 for Bimatoprost methyl

ester using positive electrospray ionization. The spectra and the proposed patterns

fragmentation, as well as a summary of the adjusted MS conditions and the

compound-specific MS-parameters, are presented in Figure 5.5 and Table 5.2.

Separation of analytes was performed on a Thenno HYPURITY C18 column

(150~2.1 mrn, 5 p) with a mobile phase consisting of 10 mmol/L ammonium

formate water-acetonitrile solution (5050, vlv) at a flow rate of 0.25 mumin. The

API4000 triple quadruple mass spectrometer was operated in multiple reactions

monitoring mode via positive electrospray ionization interface using the

transition.

Fig 5.5 Production mass spectrum of Bimatoprost ethyl ester

Table 5.2

An improved LC-MSIMS method for quantitative determination of Bimatoprost methylester

Published results [9, 101 suggest that reversed-phase HPLC is unsuitable

for analysis of the purity of Bimatoprstt and the isomers possibly present. In

accordance with literature results [1&12] for a similar class of compounds,

normal-phase c-graphy was evaluated f a separation of the isomen by the

UPLC of Bimatoprost end the using of mobile phase a homogeneous mixture of n-

Hexane, dehydrated alcohol and methanol (80: 10: 10). A typical chromatom is

shown in Fig. 5.6. It is apparent from the figure that impurities I, 11,111 and IV are

Mass spectrometer - Interface

Polarity

Scan type

Resolution

Curtain gas (CUR)

Collision gas (CAD

Ion Spray voltage (IS)

~ e r n ~ e r a k e (TEM)

Ion source gas 1 (GS 1)

Ion source gas 2 (GS 2)

Solvent split ratio

API4000

Electrospray

Polaritive

MRM

Q1-unit resolution

Q3-unit resolution

6

4500

500 'C

45

65

None

well separated fiom one another and from Bitoprost, with satisfactory peak

shapes. The result o w e d for each impurities as shown figure 5.1 0 - 5.12

Fig. 5.6 Chromatogram obtained from Bimatoprost spiked with

impurities I, 11,111 and IV

-- -- -

Fig5.8 Bimatopmt-Acid impurity

Pig.5.9 Bimatoprost-Keto impurity

Fig 5.10 Bimatoprost-Methyl ester

Fig. 5.12 Chromatogram of Bimatoprost

5.7.2 Precision

The system's precision was performed by analysing the system suitability

standard solution six t i e s . Results of Peak area of the API and the impurities are

noted. The peak area variation observed for Bimatoprost and impurities was less

than 5.0%. The results are noted comply with the acceptance criteria by indicating

acceptable precision of the system. The percentage relative standard deviation of

Peak area of six replicate injections for each impurity is 5 5.0. The Precision of

the method was determined by analyzing a sample of Birnatoprost solution spiked

with impurities at 100% of the specification limit of six replicate sample

preparations. The percentage relative standard deviation of recovery obtained for

each impurity less than or equal to 5.0. Prior to this, system suitability parameters

were calculated by injecting the system suitability solution. The %RSD was found

to be 3.01.

5.73 Specificity

Each know impurity and Bimatoprost solutions were prepared

individually at a concentration of 0.10 rnglml and a solution of all known

impurities spiked with Bimatoprost was dso prepared. A test solution of

Bimatopmst, solutions of impurities I, 11, I11 and N, and solutions of the

B i o p r o s t were spiked with the impurities. The good selectivity of the method

is ~pparent f k m Fig. 5.6.

5.7.4 Accuracy

The accuracy of the method was determined using four solutions

containing Bimatoprost spiked with the impurities at approximately LOQ, 25%,

50% 100% and 150% of the working strength of API. The percentage recovery

obtained was in the range of 100.4 - 100.6 at 80.0% to 120.0%.

5.7.5 Linearity and Range

The linearity of the method was demonstrated for Bimatoprost solutions

ranging from LOQ 20°/0, 40%, 80%, loo%, 120% and 150%. Results obtained are

shown in Table 5.3. The linearity results for Bimatoprost and impurities in the

specified concentration range were found satisfactory with a correlation

coefficient greater than 0.99.

Table 5.3

Linearity of Bimatoprost

Component

Bimatoprost

15 R-

Bimatoprost

Bimatopmst Acid

Bimatoprost Keto

Bimatoprost

methyl ester

Slope

35373637.75

29608419.44

3031 1345.27

,66859263.97

32608419.44

Intercept

697.147

-586.101

375.534

-884.5 15

-586.101

Correlation

coefficient

(R)

0.9990

0.9999

1 .MOO

0.9996

0.9999

R*

0.9979

0.9999

0.9999

0.9992

0.9999

Intercept value

w.r.to 100%

conc.std

response

1.79

-0.50

1.23

-1.34

-0.85

5.7.6 System Suitab'ily

Prepared the reference solution and the solution was analyzed six times as

per the method described.

5.7.7 Limit of detection and Limit of quantification

The l i t of detection (LOD ) is determined by calculating the signal to

noise ratio and by comparing test results from samples with known concentrations

of analyte with those of blank samples and establishing the minimum level at

which the analyte can be reliably detected. The result obtained for each impurity is

listed in Table 5.4. The limit of detection values obtained for each impurity was

within the acceptance criteria. Signal to noise ratio was about 3: 1 and the

detection was less than 0.15%.

Limit of Quantification (LOQ) values were determined from the same

experiment mentioned in the limit of detection section. The LOQ values

obtained are presented in Table 5.5 Signal to noise ratio was about 10:l and the

quantification limit is less than level of specification preferably much less.

Table 5.4

Limit of detection (LOD) for Bimatoprost and impurities

Component

Bimatoprost

15 R-

Bimatoprost

Bimatoprost Acid

Bimatoprost Keto

Bimatoprost

methyl ester

% Impurity w.r. to

working strength

0.001 1

0.0014

0.0014

0.0008

0.0014

Concentration

(mg/mJ)

0.0000221

0.0000280

0.0000275

0.0000151

0.0000280

Signal to

noise

3.5:l

2.7: 1

3.1:l

3.5:l

2.7:l

LOD (%)

0.001

0.001

0.001

0.001

0.001

Table 5 3

Limit of Quantitation for Bimatoprost and impurities

5.7.8 Robustness

Component

Bimatoprost

15 R - Bimatoprost

Bimatoprost Acid

Bimatoprost Keto

Bimatoprost methyl ester

System suitability followed by a sample analysis was run to assess if these

changes had a significant effect on the chromatography. A sample of Bimatoprost

spiked with known impurities was analyzed for verifying the level of impurities at

each variation. The retention time of all the impurities including Bimatoprost was

affected by slight variation in the flow, pH and column temperature, however the

system suitability criteria for the method was fulfilled. The number of theoretical

plates for Bimatoprost peak is not less than 3000. The resolution between the

peaks due to intermediate and Birnatoprost is not less than 2.0, The tailing factor

for Bimatoprost peak is not more than 2.0.

5.7.9 Solution stability

%Impurity w.r. to

working strength

0.004

0.005

0.005

0.003

0.005

A solution of Bimatoprost spiked with the impurities and the standard

solution stability were kept at room temperature (24-26OC) as well as in the

refrigerator at 2-8' C. The solution stability was monitored at different intervals

(Initial, 24 hours and 48 hours). No significant variation in the percentage of

impurities was observed up to 48 hours at 2-8' C for reference solution and

sample solutim The level of unknown impurity was found to inawe in the

sample solution s t o d at room kmpemtm. It is mmmendcd to keep the

Signal to noise

9.7: 1

9.51

10.O:l

9.9:l

9.5: 1

solutions at 2-8' C for analysis. The results were recorded and assigned the

stability of the solution based on the experimental data For a stable solution, the

individual impurity values are to be within *0.03 of the original value and the

total impurities are to be within kO.10 of the original value.

CONCLUSION

The method enabled baseline separation of 15(R)-Bimatoprost (impurity

I), Bimatoprost acid (impurity 11), Keto (impurity 111), and methyl ester impurity

IV, from Bimatoprost. The method is characterized by good precision, the

linearity correlation coefficients for the known impurities are better than 0.98. The

detection limits for the known impurities were below the level (0.05%).This

method commercially can be used. The validation was performed according to the

current requirements as laid down in the ICH guidelines.

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