HUMAN HEALTH • ENVIRONMENTAL HEALTH

© 2014 PerkinElmer

USP<857> for Validating

UV/Visible Spectrophotometers Steve Upstone/Helge Madsen

15th April 2016

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)

6

Why Stray Light is Important – It Affects Measurement at

Higher Absorbances

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0

True Absorbance

Me

asu

red

Ab

so

rba

nce

0.005% Stray Radiation

0.01% Stray Radiation

0.05% Stray Radiation

0.1% Stray Radiation

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

9

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 dual-

beam 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