Strict procedural controls within the pharmaceutical manufacturing industry are paramount, whether it be for raw materials testing, drug production or final quality assurance and refractometers and polarimeters play an important role in helping to ensure our medicines, whether it be for animal or human use, are safe and effective.
Refractive Index – USP <831>
When light passes from one medium to another, the speed at which it travels changes in proportion to the density of the media through which it travels. Refractive Index is therefore, the ratio of the speed of light travelling through a substance compared to vacuum (or for more practical purposes, air) and optical instruments called refractometers are used to measure refractive index. Typically refractometers use a white light source, compensated to 589nm (Sodium yellow, symbol N). The Refractive Index (R.I., symbol n) is a unique parameter of a substance and is used to help identify or quantify a substance.
To comply with pharmacopeia, refractive index has to be measured to 4 decimal places at a constant temperature, typically 20 °C (25 °C for United States Pharmacopeia, known as USP) but some pharmaceutical manufacturers stipulate a higher precision than that quoted in the norm.
There are over 80 references to refractive index measurement in pharmacopeia, most of them being part of raw materials testing.
n is the refractive index (RI)
t is the temperature in Celsius (°C)
λ is the wavelength in nanometres (nm).
The RFM970-T refractometer achieves 6-decimal resolution AND precision
Optical Rotation – USP <781>
Light also “twists” when it passes through certain “optically active” or chiral substances, which is again, a unique parameter of the medium it is passing through. Substances that cause light to twist to the left are known as Levorotatory whereas samples that cause light to twist to the right are called Dextrorotatory. “Sugars” are a good example of optically active samples; dextrose (D-glucose) causes light to twist to the right whereas fructose (sugar derived naturally in fruit) causes light to twist to the left. The type of sugar or other chiral compound used in pharmaceutical manufacture is critical to the medicine make-up and so it is crucial to ensure correctness and purity.
Polarimeters are used to measure the twist of polarised light, expressing the results in degrees angular (°A). Polarimeters are used to measure the purity of a raw material as well as testing the ratio or quality control of a blended material that contains a number of different optically active compounds such as a medicine being produced in a pharmaceutical manufacturing site in accordance with a particular recipe or monograph as it is known within pharmacopeia.
Like refractive index measurement, analysis temperatures for optical rotation are either 20 or 25 °C but as optical rotation is less affected by temperature than RI, control is to a lesser degree (typically ±0.5 °C) when compared to refractometers that must be held within ±0.05 °C to achieve high precision RI results.
Pharmacopeia quotes all polarimeter analyses as relative to the tube length, temperature, wavelength and concentration (density) of the sample being tested so that comparison between users and manufacturing sites is constant. This is known as Specific Rotation and it is calculated below.This enables the measurement of highly diluted samples or those with very a low specific rotation to be analysed in any tube length, facilitating the use of short tube lengths.
[α]tλ is the specific rotation of polarised light of wavelength λ at temperature (t) Celsius (°C)
α is the measured optical rotation
l is the length of the polarimeter tube in metres
d is the relative density of the liquid or solution at 20 °C
c is the concentration of the solute expressed in grams per mL of solution
p is the concentration of the solute expressed in gram per gram solution
Pharmaceutical measurements at wavelengths other than sodium
In some circumstances, especially when analysing optical rotation of highly diluted or scarce samples as well as those having an exceedingly low optical rotation, pharmacopeia states that measurements should be taken at a lower light frequency than the preferred sodium yellow line; such as those emitted from helium or mercury sources, including in some cases, within the ultra-violet region as this increases the sensitivity of the measurement dramatically.
Changing the ADP600 poalrimeter’s measurement wavelength is a simple two-button push operation
However, consideration must be made when calculating the specific rotation of a sample measured at lower wavelengths, especially in the UV region and across short path lengths as multiplication factors exacerbate instrument reading errors. The highest possible measurement accuracy is therefore required such as that achieved by the multiple wavelength, UV/Vis ADP600 Series polarimeters that are capable of measuring rotation to four decimal places.
Such specific rotation measurements include:
|Monograph||Wavelength||Specific rotation between|
*for Dextromethorphan Hydrobromide, actual reading must be within 1% of the reading obtained from a USP Reference Standard of the same material.
GLP & GMP within the pharmaceutical industry
Good Laboratory Practice (GLP) and Good Manufacturing Practice (GMP) also plays an important role within the pharmaceutical laboratory. A good representation of a production batch should always be taken for quality assurance testing, with the mean of the experiment being recorded. Bellingham + Stanley refractometers and polarimeters featuring a Methods system include the PHR-MEAN Method, or Pharmaceutical Mean Method that requires at least 5 samples from a single batch to be measured prior to the statistical analysis, including all readings & temperatures, the average reading and standard deviation being presented in the final report alongside the operator and batch codes being printed or output on a Secure PDF report sent via USB or LAN.
A record of instrument calibration including operator details and sample batch numbers is also an important feature of Bellingham + Stanley key line refractometers and polarimeters.
Additionally, the same key instruments feature high level security user clearance via keypad entry or by way of an RFID swipe card to access measurement, calibration and instrument configuration so that an audit trail is maintained of all instrument actions performed therefore meeting the requirements of FDA regulation 21 CFR Part 11 for electronic signature recording.
Additionally, as “general purpose laboratory instruments” not specifically designed for in vitro diagnostics, latest RFM-T Series refractometers and ADP Series polarimeters used in the production of medicines comply by exemption with EU legislation 98/79 for in vitro diagnostic equipment.
Statistical analysis reports are available from both RFM900-T and ADP600 instruments. A time delay between each individual reading from a batch of samples facilitates GLP in the pharmaceutical manufacturing industry.
Validation and verification ensures correct analysis time after time
Initial validation as part of installation (IQ/OQ/PQ) and regular verification of refractometers and polarimeters is also an important part of GLP as it ensures initial and continued instrument performance.
Although refractometer standards are cited in Pharmacopeia, these tend to be difficult samples to handle and have limited accuracy for today’s high precision refractometers such as the RFM970-T model. Instead, many pharmaceutical manufacturers now use alternative UKAS Certified Reference Materials calibrated in accordance with ISO EN 17025:2005 and traceable to NIST such as the Pharma & Chem multipack as these cover the wide range of operation needed within the pharmaceutical industry and have the added bonus of being non-hazardous in terms of usage and disposal.
Verification of polarimeters can be seen as more convenient than refractometers as inert UKAS certified Quartz Control Plates (aka QCP or Quartz Reference Standards QRS) of known rotation traceable to primary PTB standards may be used; these too being calibrated in accordance to ISO 17025:2005. Again, multi-point verification using QCP-Sets covering the measurement range of interest is ideal under GLP.
Traceable reference materials are available in convenient multi-value packs
UKAS, the United Kingdom Accreditation Service is one of the signatories of the International Laboratory Accreditation Co-operation Arrangement (ILAC) for the mutual recognition of calibration certificates and as such, UKAS certified reference materials supplied by Bellingham + Stanley are accepted by local accreditation services across the globe that are also members of ILAC and the ILAC MRA scheme.
Common features of the latest touchscreen RFM refractometers and ADP600 polarimeters benefiting the pharmaceutical manufacturing industry:
RFM900-T Series Refractometers benefit the pharmaceutical manufacturing industry:
ADP600 Series Polarimeters benefit the pharmaceutical manufacturing industry:
Real-time refractive index measurement of vaccines:
In some cases, such as during the manufacture of bird flu vaccines, refractive index measurements are required instantly so that blending or quality assurance processes are in real-time. This may be achieved by using an in-line RFM refractometer fitted with a flowcell. In order to prevent contamination of the vaccine, wetted materials including the sample chamber, gasket materials and the measuring prism itself must meet the highest hygiene (sanitary) standards, complying with FDA regulations and certified in accordance with ISO 10204.
To learn more about other Xylem products for use in the life science, medical and pharmaceutical industries, please visit www.xylemanalytics.co.uk/lifesciences