Applications of HPLC in Pharmaceutical Engineering

Qing Ye

South China Agricultural University, Material and Energy College Guangzhou, 510630, China

Keywords: HPLC, Pharmaceutical, Purification, Drug Quality, Metabolites.

Abstract: The pursuit of high quality drugs leads to increasing demand for technique with high performance in mixture

separation. Pharmaceutical industry is a field requiring fine separation technology. High performance liquid

chromatography (HPLC) is commonly seen in pharmaceutical study and manufacture in separation processes

for diverse aims. In this paper, the related information from some databases are collected and summarized to

discuss three major applications including the purification of raw material, quality analysis of drug product

and assay of metabolites. Evidences show that HPLC has broad purpose by pharmaceutical engineering. This

paper is intended to offer an overview on applications of HPLC in pharmaceutical engineering and provide

ideas for improvements of technique with new methods.

1 INTRODUCTION

Chromatography has been developed a lot in the last

hundred years since the initial separating the plant

pigments. For the over decades, its analytical ability

have been greatly improved by the advance of

equipment. High performance liquid chromatography

(HPLC) is a rapid separation instrument commonly

used in pharmaceutical engineering, which is based

on the principle that different materials own different

distribution coefficients in a certain stationary phase

and mobile phase.

Pharmaceutical engineering is a field has high

requirement for separation. The fast running speed of

HPLC maks it one of the most applied tools in

substances separation and real-time detection in

several important segments pharmaceutical industry.

Connected with detective devices, it can help to

detect residual substances, determine the drug

content, analyse drug metabolism and so on (Ahuja

2017). HPLC plays an important role in the producing

process of those products with high purity

requirement. It is applied in the purification of

alkaloid and peptide which takes a quite low

proportion in the mixture (Wu et al. 2013). Besides,

it is also widely applied in drug metabolism analysis

for its high separation efficiency.

Recently, more and more specific HPLC methods

are developed for the research on certain drug or

preparation, which helps people to know better about

the drug’s properties and the improve its quality.

Therefore, the following paragraphs will focus on

three major applications of HPLC: purification,

quality analysis and metabolism analysis.

2 RELEVANT ANALYSIS ON

HPLC

2.1 HPLC for Drug Purification

A considerable part of the existing drugs come from

natural products of plants or microorganism.

Therefore, purification with high efficacy is an

indispensable stage during drug discovery and

manufacture. HPLC displays a good performance in

separating and purifying substances.

One of the problems in deriving natural product is

that the content of the active ingredient is very low

and the components are complicated, making

separation difficult. Fortunately, HPLC helps this

process easier. Liao X set up a method to determine

the active components of Osmanthus fragrans roots

by HPLC-MS/MS (Liao et al. 2021). In this

experiment, 36 compounds were detected in the

separation and one of them was newly found in the

work. Tandem mass spectrometer (MS/MS) helped to

measure the relative molecular mass of each

component segregated by HPLC, providing key

information for chemical structures. Hong Y and his

partners also used a similar method (HPLC-MS/MS)

962

Ye, Q.

Applications of HPLC in Pharmaceutical Engineering.

DOI: 10.5220/0011374700003443

In Proceedings of the 4th Inter national Conference on Biomedical Engineer ing and Bioinformatics (ICBEB 2022), pages 962-967

ISBN: 978-989-758-595-1

to determine the bioactive ingredients of Cercis

chinensis Bunge fruits (Hong et al. 2020). Gallic acid

was found to account for the largest proportion. This

has not been reported before, indicating some new

medicinal value of the fruits.

High throughput purification is demanded in

pharmaceutical industry. However, it’s not easy to

realize that in the production of antibody, cytokine

and other kinds of peptide drug. A large scale HPLC

system used for purifying IgGs was validated by

Schmitz S and his team (Schmitz et al. 2019). Three

running pumps were paralleled and work

simultaneously in this method to ensure the rapid

removal of aggregates and endotoxins. They also

used the autosampler to make the process automatic

and enable continuous manufacturing.

Besides changing the parameter of

chromatography, modifying the target product also

makes purification efficient. Tag-assisted technology

is quite useful in peptide drugs. The filler of column

binds the corresponding groups that can specifically

combine the tag of target protein. By this process,

separation with high resolution can be realized by

specific binding and elution. To simplify the

procedures, the self-cleavable tags have been

developed as well as the aggregating tags (Mmg et al.

2020). Such tags are intended to reduce the

pretreatment or post processing.

High purity and high production are two goals that

manufacturer wants to achieve. But these two

objectives are often contradictory due to the limit of

current manufacturing technique. Luca C. D. and her

team have established a multicolumn continuous

chromatography to solve the problem (Luca et al.

2020). Different from a classical forwardcurrent

chromatography, the method employed

countercurrent solvent to purify the products without

a solid filler column. An automatic internal recycling

was set in the system to increase production with

acceptable purity.

2.2 HPLC for Drug Quality

Drug quality inspection is a necessary step to ensure

the safety of preparations after production. Quality

and impurity determination are two general test items

for most preparations. HPLC is quite practical in

preparation analysis due to high resolution and

rapidity.

HPLC can be used for the determination of drugs

with similar chemical structures when a suitable

mobile phase and proper elution gradient are set.

Trifluoperazine and prochlorperazine are both

phenothiazine derivatives with only one different

group on benzene ring. According to Dhabab

(Dhabab et al. 2013), a determination method for

these two drugs was conducted by using reverse

phase HPLC (C18 column, acetonitrile as mobile

phase, chlorperazine hydrochloride as internal

standard), showing a good separation and acceptable

accuracy in quality (recovery percent is higher than

96%).

By applying a chiral stationary phase, HPLC can

be used to analyse the enantiomeric purity of certain

drugs. Sertaline is an antidepression drug with two

stereogenic centers. Ara B et al. have demonstrated a

reverse phase method for determining enantiomeric

purity of sertraline by using Chiralpak IG-3 column

with acetonitrile-water-DEA mixtures (75:25:0.1,

V/V/V) as mobile phase (Ara et al. 2020). The result

showed that it could recognize another two

stereoisomers of sertraline well within 15 min by a

single-run approach, reducing the time consumption

and expenditure of existing method.

HPLC is also applied in simultaneous estimation

for compound preparations containing more than one

active pharmaceutical ingredients. A simple, rapid

HPLC method for estimation of three antivirus drugs

(Lamivudine, Tenofovir, and Dolutegravir) is

developed by Rao N M et al (Rao et al. 2015). They

adopt a Inertsil ODS-3V C18 column with mobile

phase of mobile preparation A (mixture of potassium

dihydrogen orthophosphate buffer and methanol) and

B (mixture of orthophosphoric acid and acetonitrile).

These drugs can be separated within 14 mins and high

mean percent of recovery by different added amount

(all are over 99%), displaying a good applicability of

this method.

Test of drug degradation is a necessary step to

ensure its quality. HPLC can be applied to analyse the

degradation substances of the drug under certain

stress. Bisht R et al. have developed a reverse phase

HPLC method connected with UV detector for

determining the degradation of connexin43 mimetic

peptide (Bisht et al. 2017). It presented good linearity

between the concentration of 0.9-250 μg. The result

showed that the peptide, which helps to treat

inflammation, was sensitive to temperature and pH.

In addition, the analysis of excipient takes

advantage of some detection technology combined

with HPLC. Liposome is a good coating to improve

drug’s hydrophilicity. But one technical difficulty is

how to determine the uniformity of each liposme as

well as the drug inside it. Langer C and R Süss have

created an HPLC method applied in a range of

liposome drugs using diode array detector (DAD) for

qualitative detection and corona charged aerosol

detector (CAD) for quantitative detection (Langer et

Applications of HPLC in Pharmaceutical Engineering

963

al. 2021). The benefit of CAD is the non-reliance on

the structures of substance. Instead, it depends on the

number of charged atomized particles. Therefore, it

can detect both the drug and liposomes after they

were separated by HPLC. The method has been also

validated for cyclodextrin coated drugs.

2.3 HPLC for Drug Metabolism

Drug metabolism analysis is an essential part during

the study of pharmacokinetics. One of the common

features of biological samples is the complex

composition. So the technique for detection in animal

experiment and clinical trial needs to be precise and

effective. HPLC plays an important role in this part.

Plasma is the most commonly used biological

sample. Sws A et al. have established an HPLC

method combined with fluorescence detector (FLD)

for determination of the metabolism of alpelisib in rat

plasma (Sws et al. 2020). FLD is suitable for

molecules which can emit fluorescence under certain

wavelength radiation. Alpelisib, an antibreastcancer

drug, has two aromatic rings which allow it to have a

significant response in test. The result presented that

the drug kept stable in blood for 24 hours.

Prodrug is an effective way to improve the

bioavailability. Finding out the metabolic pathway is

a significant step to ensure the safety of the drug.

Nobilis M and his team developed a method for

determination of the metabolites of new nabumetone

(a type of anti-inflammatory prodrugs) (Nobilis et al.

2013). They first used liquid-liquid-extraciton to

collect the compounds in liver microsomal fractions

(including human tissue and rat tissue), and then

employed HPLC with photo-diode array and tandem

mass spectrometer to analyse the metabolites. 3-

hydroxy nabumetone was found and it was inferred

to be the first metabolite of new nabumetone after a

further study on its biotransformation.

In order to make the detection comprehensive,

radioactive elements are used to track the distribution

and metabolism of drugs in the body. Accordingly,

the radio-HPLC is established for this kind of test.

Gaudin A et al. have developed a method to detect the

squalenoyl adenosine nanoparticles (radio-labeled

with 3H and 14C) in mice (Gaudin et al. 2015). In this

paper, the nucleoside is covalented to squalene to

extend its half-life. Mice plasma was used in the

stability determination of the drug in vitro and it was

separated by the HPLC and then detected by a

radioactive element detector. The result indicated that

the prodrug was able to persist for 1 hour and mainly

absorbed by the liver and spleen.

Multidimensional chromatography is a

technology that combines columns with different

selectivity or several types of chromatography

methods to improve the separation effect as much as

possible. Roberta K et al. have established a method

for the determination of albendazole metabolites in

microsomal fractions of mice liver (Kátia et al. 2013).

A chiral 2D-HPLC was employed in this work where

a bovine serum albumin column was for sample

clean-up and the other column for chiral resolution.

Result showed that sulfonylation to albendazole

occurred in liver tissue.

3 THE RESEARCH SITUATION

OF HPLC IN

PHARMACEUTICAL FIELD

Published papers in this field reflects the

development of the technqiue. By using the

visualized analysis of the database CNKI, the author

input key words “HPLC drug” and derived the overall

trend of the applications of HPLC in pharmaceutical

field in China of recent years.

Figure 1: The annual published trend versus year

Figure 1 presents the change of the number

relevant published papers. A huge growth of the

number for over 1000 is seen from 2016 to 2017.

Then the quantity has dropped a bit in the following

-500

500

1000

1500

2015 2016 2017 2018 2019 2020 2021

The Number of

Published

Papers

Year

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

964

years but has increased again since 2020. This

indicates that more attention has been paid to the

study of HPLC in the field since 2016.

Figure 2: The distribution of major topics of the papers.

Figure 2 shows the different topics of the relevant

papers and their respective quantities. Study on

pharmacokinetics has taken up the highest number

with 242 articles in the recent 5 years, while the

HPLC method study ranked the second place with

close numbers of simultaneous determination and

chemical components. It can be inferred that HPLC

mainly serves in medicine analysis and the study on

purification for manufacture is rare.

Table 1: Application of IC-FLD/UV for the determination of different drugs.

Researches also concern about the comparison of

the application of HPLC by different drugs. Ion

chromatography (a type of HPLC with ion exchange

resin as solid phase) is used to analyse ionized

substance and table 1 gives figures of the

determination of several drugs applying IC

(Separovic et al. 2018). As it’s shown, FLD and UV

are commonly used as detectors, though the specific

condition of the chromatography could vary a lot. The

following data of each drug presented different

ranges of LOD and LOQ, indicating that the

performance of IC might be not universally

referential, while the same thing happened by

recovery with fluctuation of different degree. It might

be even doubtful whether HPLC is suitable for a

certain drug. Hence, a precise standard of HPLC

applicability for drugs should be defined to provide

more reference for quality analysis.

4 CONCLUSIONS

Overall, HPLC possesses good separation efficiency,

universal applications and easy operation, thus

making it practical in many fields of pharmaceutical

engineering. However, some disadvantages of HPLC

should not be neglected. Measurement uncertainty of

the equipment is a factor influencing the quality of

drug products. According to a study on HPLC

determination of amoxicillin tablets (Muhammad et

al. 2021), it has been found that the uncertainty of the

whole process exceeded the recommended value and

this has forced the manufacturer to take a higher risk.

This suggests that addictied measures should be taken

to check the accuracy during the process. Besides,

environmental burden and human health must be

considered since the organic solvents have a long-

term effect by accumulation. Recently, more and

more modified method using green solvents like

242

150

145

143

32 32

27 27

100

150

200

250

pharmacokin…

HPLC…

simultaneous…

chemical…

HPLC-MS/MS

fingerprint

biological…

drug…

primary…

liposome

quality…

impurity

in vitro and…

preparation…

antitumor…

nanoparticles

quality control

RP-HPLC

entrapment…

The Number of

Papers

Main topics of the papers

Applications of HPLC in Pharmaceutical Engineering

965

ethanol and acetic acid or even free solvent are being

proposed (Mya et al. 2020, Mikhail et al. 2021). It is

expected that more green and efficient methods will

be found and established.

Although some problems exist in the HPLC

method for pharmaceutical engineering, it still

remains the necessary technique in substance

purification, drug quality analysis and metabolism

assay. Wide range of application including chemical

drug, peptide drug, chiral drug and so on, makes it

adopted nearly throughout the whole process.

Overall, it is anticipated that more improved methods

will be put forward to overcome the shortcomings.

The findings above is aimed to provide some basic

facts of the applications of HPLC in pharmaceutical

engineering and more innovation of the method is

expected to be inspired.

ACKNOWLEDGMENTS

The author thanks Professor Axel Zeitler for giving

lectures on introduction to pharmaceutical

engineering. The author also thanks the instructor

Cuihong Wang for her advice on this paper.

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