USP<857> for Validating UV/Visible Spectrophotometers Steve Upstone/Helge Madsen 15th April 2016
HUM AN HEALT H • ENVIRONM E N TAL HEALT H © 2014 PerkinElmer
USP<857>
• Introduced on 1st May 2015 • Mandatory from 1st May 2016 (USP numbers <1000 are mandatory, > 1000, advisory) • Covers validation of UV/Visible Spectrophotometers • Major change is the new stray light tests. Other checks similar to existing Ph Eur (European Pharmacopoeia)
USP <857> Document
What Testing is Covered by USP<857>? • Control of Wavelengths ◦ D2 or Hg emission lines ◦ Rare earth solutions (eg HoClO2, didymium) ◦ Rare earth glasses (Holmium, didymium)
• Control of Absorbance ◦ Potassium Dichromate (235, 257, 313 and 350nm) – 430nm not mandated ◦ ND glass filters (eg NIST 930e, 1930 or 2930 grey-glass)
• Stray Light ◦ ◦ ◦ ◦
KCl (12g/L aqueous) 190-205nm region NaI (10g/L aqueous) 210-259nm region Acetone (pure) 250-320nm region NaNO2 (50g/L aqueous) 300-385nm region
• Resolution ◦ Toluene in hexane (0.02% v/v solution)
What Testing is Covered by USP<857>? • Control of Wavelengths ◦ D2 or Hg emission lines ◦ Rare earth solutions (eg HoClO2, didymium) ◦ Rare earth glasses (Holmium, didymium)
• Control of Absorbance ◦ Potassium Dichromate (235, 257, 313 and 350nm) – 430nm not mandated ◦ ND glass filters (eg NIST 930e, 1930 or 2930 grey-glass)
• Stray Light ◦ ◦ ◦ ◦
KCl (12g/L aqueous) 190-205nm region NaI (10g/L aqueous) 210-259nm region Acetone (pure) 250-320nm region NaNO2 (50g/L aqueous) 300-385nm region
• Resolution ◦ Toluene in hexane (0.020% v/v solution)
Why Stray Light is Important – It Affects Measurement at Higher Absorbances
5.0
Measured Absorbance
4.5 4.0 3.5 3.0 2.5 2.0
0.005% Stray Radiation 0.01% Stray Radiation 0.05% Stray Radiation 0.1% Stray Radiation
1.5 1.0 0.5 0.0 0.0
1.0
2.0
3.0
4.0
True Absorbance
5.0
6.0
7.0
Stray Light – How it is Manifested
How Stray Light Has Been Measured in the Past • Comparison of trace in transmittance with a blocked beam (ASTM method using NaI and NaNO2) • Measurement of absorbance at a specified test point (eg Ph Eur test using 12g/L aqueous KCl at 200nm or 198nm) – should exceed a minimum value (eg 2A)
Ph Eur Stray Light Test
ASTM Stray Light Methodology
• ASTM methodology measures stray light (in transmittance compared to a block beam) using NaI, NaNO2 and acetone
USP<857> Methodology for Stray Light
• Based on 1982 paper of Mielenz et al. (Appl Opt. 1982 Sep 15;21(18):3354-6. doi: 10.1364/AO.21.003354.)
• Method developed around PMT/ chopper based instrumentation (only type that was around in 1982) so some features of the test are now superfluous and could cause issues with current dualbeam and PDA instruments • Requires four standards (KCl, NaI, NaNO2, Acetone) in 10mm sealed cuvettes – together with 5mm sealed cuvettes for the reference channel
Methodology Developed on Double Beam Spectrophotometer • Original 1982 Mielenz paper based on chopper-based double beam instrument where beam is multiplexed between sample and reference position 25 times a second
Principle of Reference Beam Attenuation for Double Beam Instruments The top picture shows the system what happens in a double beam instrument when a highly absorbing sample is measured. The energy difference between the front and rear beam is too great and the system becomes noisy By attenuating the reference beam, the energy difference becomes less and the noise is eliminated
USP<857> Standards Set
What is Being Measured – Measurement of the difference between a 10mm and 5mm NaI cuvette
What the Result Looks Like - Stray Light Measurement (NaI)
Different Instruments Give Different “Peak” Locations
Data source: Starna Catalog
Stray Light Calculation
• Maximum absorbance (A) is recorded • Stray Light Defined by
• 𝑆𝑡𝑟𝑎𝑦 𝐿𝑖𝑔ℎ𝑡 𝑠 = 0.25 ∗ 10(−2∗𝐴) • A must be >=0.7 and S =< 0.01 to pass
Typical Lambda 25/35 Values (1nm slit)
Test
Stray Light
KCl
0.0048
NaI
0.0016
Acetone
0.0014
NaNO2
0.0028
Potential Issues • Instrument has to have ability and sensitivity to measure close to 4AU – most solid state instruments can measure up to around 3AU • Using the 5mm cuvette as a reference beam attenuator is irrelevant to non-PMT/chopper instruments. It actually makes life worse for dual beam and photodiode array spectrophotometers • Customers may mistake the maximum as being a “peak” and attempt to use it as a way of calibrating wavelengths or indicate that there is a “problem” with the instrument if it varies from the position found on the standards certificate. • Not all instruments can measure above 3AU so the norm recommends attenuation during the autozero phase • The norm is ambiguous regarding instruments with only one sample position (ie no reference position)
Bag of nails…..?
• Definition taken from ”The 1811 Dictionary of the Vulgar Tongue”, originally by Francis Grose
• ”He squints like a bag of nails…” ie his eyes are directed as many ways as the points of a bag of nails
Compatibility with New PerkinElmer UOQ
New PerkinElmer Instruments for Pharmaceutical Analysis
LAMBDA 365
LAMBDA 465 PDA
