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HPLC analysis according to USP and Ph. Eur.

HPLC analysis by drug: USP and Ph. Eur.

In the pharmaceutical industry, the regulations on the respective pharmacopoeia are of great importance. The pharmacopoeias with the highest world-wide relevance are USP (United States Pharmacopoeia) , Ph. Eur. (European Pharmacopoeia) and JP (Japanese Pharmacopoeia). The ICH (the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use) is also increasingly working towards an international standardization of the above mentioned regulations.

The objective of these pharmacopoeias is to ensure a consistent and uniform quality of the starting materials and medicinal products and thus a safe application for the patient by legally binding regulations.

The monograph part of the respective pharmacopoeia is on this concern especially relevant for the analysis. Among other things this part specifies on the corresponding methods an active substance in a drug is analyzed.  Many of these methods are also based on high-pressure liquid chromatography (HPLC). The monographs describe the stationary and mobile phases, as well as the further chromatographic conditions, such as column temperature, injection volume, etc. Even the deviations allowed by the methods, without a revalidation of the whole method being necessary, are controlled by the pharmacopoeia.

This page is intended to give an overview of the general regulations of the most important medicines for the German-speaking area, the Ph. Eur. and the USP concerning HPLC analysis.

Requirements of Ph. Eur. and USP

HPLC columns

Both Ph. Eur. And USP specify the stationary phase according to the "chemistry" of the packing material. For example, a column with the specification USP L1 corresponds to a reversed-phase column based on octadecylsilane (ODS), which has a particle size of 1.5-10 μm and is either chemically bound to porous or non-porous silica or to ceramic microparticles or else is present in monolithic form. Due to the continuous development in HPLC analysis, the list of column specifications has been and is being expanded over and over again. Currently, the USP contains more than 70 different filling materials (see below).

In the monographs, the analytical column is given only by this classification. In order for the method to remain compliant with the respective monograph, the specification of the column must be complied with and must not be changed. Also the dimensions of the HPLC column which have to be used are clearyl defined. However, Ph. Eur. And USP allow a certain scope for method optimization (see Permitted deviations according to Ph. Eur. And USP).

For the practical implementation of the analysis, this means that only the column specification has to be maintained. The choice of dimensions comes with a certain scope of freedom. Further parameters of the stationary phase such as carbon load or endcapping of the free silanol groups are not specified in the monographs. This allows the user to choose an analytical column according to his requirements from the plurality of available columns of a category.

In order to facilitate the selection of a chromatographic column, many manufacturers offer the possibility to narrow down their portfolio according to the USP L number. An alternative to the search for suitable stationary phases across different manufacturers is provided by the column configurator. Here, the USP L number can be selectively chosen and the suitable columns of the current manufacturers can be compared with one another .

Mobile phase

The mobile phase as a counterpart to the stationary phase is also defined by the monograph. Changes for method optimization concerning their composition are allowed, if they are within the permissible range (see permitted deviations according to Ph. Eur. And USP).

Chromatographic conditions

Other chromatographic conditions include flow rate, column temperature, and injection volume. Due to the interplay of the HPLC column with its dimensions, deviations from the conditions set out in the monograph are also allowed here (see permitted deviations according to Ph. Eur. And USP ).

Detection is also part of the analysis. A UV / Vis detector or DAD (diode array detector) is most frequently used for this purpose. In these, the detection is based on the light absorption of the substances to be investigated at certain wavelengths. A change in the wavelengths indicated in the monographs is therefore not permitted.

Permissible deviations according to Ph. Eur. And USP

Current status

Both Ph. Eur. and USP permit the modification of the methods listed in the monographs. If the modification of the parameters is carried out within the permissible limits, proof of system suitability is sufficient, revalidation of the modified method is not necessary. The goal of a possible modification is, in principle, the optimization of a method by which the requirements of the system are fulfilled.

According to the current state, the following modifications are allowed:

Property

USP General Chapter 621

Ph.Eur.  General Chapter 2.2.46

Column length

±70%

±70%

Particle size

Reduction by 50%

No increase

Reduction by 50%

No increase

Internal diameter

Can be adapted as long as the linear flow velocity remains the same

±25%

Flow rate

±50% or more, provided the linear flow velocity remains the same

±50%

Column temperature

±10 °C

±10 °C, maximum 60 °C

Injection volume

Reduction allowed as far as precision and detection limit acceptable.

No increase.

Reduction allowed as far as precision and detection limit acceptable.

No increase.

pH of the mobile phase

±0.2 units

±0.2 units (±1% for neutral substances)

Salt concentration of the buffer

±10%, as far as the allowed change in pH value

±10%

Composition of mobile phase

Minor components ±30%, if not more than ±10% absolute*

Minor components ±30%,if not more than ±2% absolute (greater value accepted)

  • *For gradient separation, a change of the mobile phase is not recommended. Here, another column of the same specification should be chosen or an adaption of the dead volume or the isocratic stage at the beginning of the gradient.

For classical HPLC analysis, the user has been given a generous amount of freedom to optimize the method for its conditions without revalidation of the entire method being necessary.

Due to the development of UHPLC (Ultra High Performance Liquid Chromatography) and the associated new column materials ("sub-2 μm"), however, the limits of permissible deviations were quickly reached. In order to be able to use the UHPLC conforming to the monographs, a revision of the permissible modifications was necessary.

Planned change

This was followed by a revision of Chapter 621 of the USP (USP35-NF30), which takes account of developments in recent years in the field of HPLC analysis. The following changes were made:

Property

USP General Chapter 621

Revision USP 621 (USP35-NF30)

Column length

±70%

Column length and particle sizes can be altered if their quotient remains equal to ±25%.

Particle size

Reduction by 50%

No increase

Alternatively, other combinations of column length and particle size are possible as far as the soil number dies not decrease more than 25% and 50% increase.*

Internal diameter

Can be adapted as long as the linear flow velocity remains the same

Can be adapted as long as the linear flow velocity remains the same.

Flow rate

±50% or more, provided the linear flow velocity remains the same

Adjustment according to the column length, the internal diameter and the particle size.*

Column temperature

±10 °C

±10 °C**

Injection volume

Reduction allowed as far as precision and detection limit acceptable.

No increase.

Adaption in both directions, as far as accuracy and detection limit are acceptable.**

pH of the mobile phase

±0.2 units

±0.2 units**

Salt concentration of the buffer

±10%, as far as the allowed change in pH value

±10%, as far as permissble pH changes**

Composition of mobile phase

Minor components ±30%, if not more than ±10% absolute*

Minor components ±30%, if not more than ±10% absolute**

  • *Exclusively for isocratic separation
  • **Also for gradient separation

As an example of the changes of isocratic methods made possible by the revision, USP35-NF30 lists the combinations which will be p ermissible for the same number of floors.

Length [mm]

Internal diameter

[mm]

Particle size

[µm]

Quotient Length/particle size

Flow

[ml/min]

Number of floors

Pressure

Running time

[min]

250

4.6

10

25000

0.5

0.8

0.2

3.3

150

4.6

5

30000

1.0

1.0

1.0

1.0

150

2.1

5

30000

0.2

1.0

1.0

1.0

100

4.6

3.5

28600

1.4

1.0

1.9

0.5

100

2.1

3.5

28600

0.3

1.0

1.9

0.5

75

4.6

2.5

30000

2.0

1.0

4.0

0.3

75

2.1

2.5

30000

0.4

1.0

4.0

0.3

50

4.6

1.7

29400

2.9

1.0

8.5

0.1

50

2.1

1.7

29400

0.6

1.0

8.5

0.1

The table shows the potential of the revised targets. These are taken into account by the greater flexibility in the selection of the column dimensions of the further development of the HPLC to the UHPLC. After their implementation, method transfer to sub-2 μm materials will also be permitted in future - and thus the time and cost-efficient use of UHPLC will also be possible.

USP L Column Listing

L1

Octadecyl silicon (C18-Sil) chemically bonded to porous or non-porous silica or ceramic microparticles (1.5 to 10 μm diameter) or monolithic silica rods.

L2

Solid spherical cores coated with octadecyl-silicon (C18-Si) binding silica gel with controlled surface porosity (30 to 50 μm diameter).

L3

Porous silica particles (1.5 to 10 μm diameter) or monolithic silica rods.

L4

Silica gel with controlled surface porosity bound to a solid spherical core (30 to 50 μm diameter).

L5

Aluminum with controlled surface porosity bound to a solid spherical core (30 to 50 μm diameter).

L6

Strong cation exchanger packed with a sulfonated fluorocarbon polymer bound to a solid spherical core (30 to 50 μm diameter).

L7

Octadecyl silicon (C18-Sil) chemically bonded to completely or superficially porous silica particles (1.5 to 10 μm diameter) or monolithic silica rods.

L8

A monomolecular layer of aminopropyl silicon chemically bonded to completely porous silica gel as carrier material (1.5 to 10 μm diameter).

L9

Irregularly shaped or spherical, completely porous silica gel coated with chemically bound, acidic cation exchangers (3 to 10 μm diameter).

L10

Nitrile groups chemically bound to porous silica particles (1.5 to 10 μm diameter).

L11

Phenyl groups chemically bound to porous silica particles (1.5 to 10 μm diameter).

L12

A strong anion exchanger prepared from quaternary amines chemically bound to a solid spherical silicane (30 to 50 μm diameter).

L13

Trimethylsilane chemically bound to porous silica particles (3 to 10 μm diameter).

L14

Silica gel coated with chemically bound, strongly basic, quaternary ammonium anion exchangers (5 to 10 μm diameter).

L15

Hexylsilane chemically bound to completely porous silica particles (3 to 10 μm diameter).

L16

Dimethylsilane chemically bound to porous silica particles (5 to 10 μm diameter).

L17

Strong cation exchange resin consisting of sulfonated cross-linked styrene-divinylbenzene copolymers in hydrogen form (6 to 12 μm diameter).

L18

Amino and cyano groups chemically bound to porous silica particles (3 to 10 μm diameter).

L19

Strong cation exchange resin consisting of sulfonated cross-linked styrene-divinylbenzene copolymers in calcium form (about 9 μm diameter).

L20

Dihydroxypropane groups chemically bonded to porous silica or hybrid particles (1.5 to 10 μm diameter).

L21

A solid, spherical styrene-divinylbenzene copolymer (3 to 10 μm diameter).

L22

A cation exchange resin made of porous polystyrene gel having sulfuric acid groups (about 10 μm in diameter).

L23

An anion exchange resin made of porous polymethacrylate or polyacrylate gel having quaternary ammonium groups (7 to 12 μm diameter).

L24

A semi-solid, hydrophilic gel consisting of vinyl polymers with several hydroxyl groups in a surface matrix (32 - 63 μm diameter).

L25

A packing material capable of separating components having a molecular weight of 100 to 5000 (defined by polyethylene oxide), applicable to neutral, anionic or cationic, water-soluble polymers. A polymethacrylate resin base crosslinked with polyhydroxylated ether. The surface has some remaining carboxyl groups.

L26

Butylsilane chemically bonded to completely porous silica particles (1.5 to 10 μm diameter).

L27

Porous silica particles (30 to 50 μm diameter).

L28

A versatile carrier material consisting of high-purity, spherical silica substrate (100 Å), to which anion exchangers are bound to ensure amino functionality in addition to the conventional C8 reversed phase functionality.

L29

Gamma aluminum, reversed phase, low carbon weight fraction, spherical polybutadiene particles based on aluminum with a pore volume of 80 Å (5 μm diameter).

L30

Ethylsilane chemically bonded to completely porous silica particles (3 to 10 μm diameter).

L31

A hydroxide-selective, strong anion-exchange resin with quaternary amines bound to latex particles, joined to 8.5 μm-sized macroporous particles with a pore size of 2000 Å. Prepared from ethyl vinyl benzene crosslinked with 55% divinylbenzene.

L32

A chiral ligand exchange resin. L-proline copper complex covanlent bound to irregularly shaped silica particles (5 to 10 μm diameter).

L33

A packing material with the possibility of separating dextrans from 4,000 to 500,000 Da. The spherical, silica-based cores are optimized for high pH stability.

L34

Strong cation exchange resin consisting of sulfonated crosslinked styrene-divinylbenzene copolymer in lead form (about 7 to 9 μm diameter).

L35

A zirconium-stabilized, spherical silica packing material having a hydrophilic, monomolecular (diol) type and pore size of 150 Å.

L36

A 3,5-dinitrobenzoyl derivative of L-phenylglycine covalently bound to 5 μm aminopropy silicon.

L37

A polymethacrylate gel with the possibility of dissolving proteins with a molecular weight of 2,000 to 40,000 Da.

L38

A methacrylate-based size exclusion package for water-soluble samples.

L39

A hydrophilic polyhydroxymethacrylate gel of wholly porous, spherical resin.

L40

Cellulose tris-3,5-dimethylphenylcarbamate coated porous silica particles (5 to 20 μm diameter).

L41

Immobilized α1-acid glycoprotein on spherical silica particles (5 μm diameter).

L42

Octylsilane (C8-sil) and octadecylsilane groups (C18-sil) chemically bonded to porous silica particles (5 μm diameter).

L43

Pentafluorophenyl groups chemically bonded to silica particles by means of a propyl spacer (5 to 10 μm diameter).

L44

A multifunctional support material consisting of high-purity, spherical silica substrate (60 Å), to which a cation exchanger is attached having a sulfuric acid functionality in addition to the conventional C8 reversed phase functionality.

L45

Bety-cyclodextrin bound to porous silica particles (5 to 10 μm diameter).

L46

Polystyrene / divinylbenzene substrate agglomerated with latex particles having quaternary amino functionalities (about 9 to 11 μm diameter).

L47

Microporous anion-exchange substrate with high capacity, fully functionalized with trimethylamine groups (8 μm diameter).

L48

Sulfonized cross-linked polystyrene having an outer layer of porous anion-exchange microparticles (10 to 15 μm in diameter).

L49

A reversed phase packing material consisting of a thin layer of polybutadiene on spherical, porous zirconium particles (3 to 10 μm).

L50

Multifunctional resin with reversed phase retention and strong anion exchange functionalities. The resin consists of 55% ethyl vinyl benzene crosslinked with divinylbenzene (3 to 15 μm diameter) and a surface area of ‚Äč‚Äčless than 350 g per m2. The substrate is coated with latex particles having quaternary ammonium groups.

L51

Amolytic tris-3,5-dimethylphenylcarbamate, porous, spherical silica particles (5 to 10 μm diameter).

L52

A strong cation exchange resin made from porous silica having sulfopropyl groups (5 to 10 μm diameter).

L53

A weak cation exchange resin consisting of ethyl vinyl benzene crosslinked with divinylbenzene (3 to 15 μm diameter) to 55%. The substrate is surface-refined with monomers having carbonic and / or phosphoric acid groups. The capacity is at least 500 μEq / column.

L54

A size exclusion material made of covalently bound dextran on highly crosslinked, porous agarose particles (about 13 μm diameter).

L55

A strong cation exchange resin made of porous silica coated with polybutadiene-malonic acid copolymer (about 5 μm in diameter).

L56

Propylsilane chemically bonded to completely porous silica particles (3 to 10 μm diameter).

L57

A protein for chiral detection chemically bound to silica particles with a pore size of 120 Å (approx. 5 μm diameter).

L58

Strong cation exchange resin consisting of sulfonated cross-linked styrene-divinylbenzene copolymer in sodium form (about 6 to 30 μm diameter).

L59

Packing material for the size exclusion of proteins ranging from 5 to 7,000 kDa. It is spherical (1.5 to 10 μm diameter) based on silica or a hydrophilic coating hybrid.

L60

Spherical, porous silica gel with covalently bonded alkylamide groups on the surface (endcapped, <10 μm diameter).

L61

A hydroxide-selective, strong anion exchange resin consisting of highly cross-linked microporous particles (13 μm diameter) having a pore size of less than 10 Å. The material used is an ethyl vinylbenzene crosslinked with 55% crosslinked divinylbenzene with latex coating consisting of 85 nm microparticles with quaternary alcohol ammonium ions (6%).

L62

C30 silane chemically bound to completely porous, spherical silica particles (3 to 15 μm diameter).

L63

The glycopeptide teicoplanin bound to spherical silica having a pore size of 100 Å by several covalent bonds.

L64

Highly basic anion exchange resin consisting of 8% cross-linked styrene-divinylbenzene copolymer having a quaternary ammonium group in chlorid form (45 to 180 μm diameter).

L65

Strongly acidic cation exchange resin consisting of 8% cross-linked styrene-divinylbenzene copolymer having a sulfuric acid group in hydrogen form (63 to 250 μm diameter).

L66

A silica gel substrate coated with a crown ether (5 μm diameter). The active side is a (S) -18-crown-6-ether.

L67

A porous vinyl alcohol copolymer having a C18 alkyl group bonded to the hydroxyl group of the polymer (2 to 10 μm diameter).

L68

A spherical, porous silica (<10 μm diameter). The surface is coated with covalently bonded alkylamino groups (no endcapping).

L69

Ethyl vinyl benzene / divinyl benzene substrate agglomerated with latex particles having quaternary amino functionalities (about 6.5 μm diameter).

L70

Silica coated with cellulose tris (phenyl carbamate) (5 μm diameter).

L71

A solid, spherical polymethacrylet (4 to 6 μm diameter).

L72

(S) -phenylglycine and 3,5-dinitroanaline-urea are covalently bound to silica.

L73

Solid, spherical polyvinylbenzene particles (5 to 10 μm diameter).

L74

A strong anion-exchange resin consisting of highly cross-linked nuclei. The cores are composed of macroporous particles (7 μm diameter) with a pore size of 100 Å. 55% crosslinked divinylbenzene and an anion-exchange layer bonded to the surface are functionalized with quaternary alkyl-ammoinium ions.

L75

A protein for chiral recognition, BSA, chemically bound to silica particles with a pore size of 300 Å (about 7 μm diameter).

The suitable columns for USP can be found in our HPLC column configurator .