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3.4 Crude Protein by Near Infrared Reflectance Spectroscopy

Reference:
Fiber (Acid Detergent) and Protein (Crude) in Animal Feed and Forages: Near-infrared Reflectance Spectroscopic Method. (989.03) Official Methods of Analysis. 1990. Association of Official Analytical Chemists. 15th Edition.

Martin, G.C., J.S. Shenk, and F.E. Barton II. 1989. Near Infrared Reflectance Spectroscopy (NIRS): Analysis of Forage Quality. United States Department of Agriculture, Agricultural Research Service. Agricultural Handbook No. 643.

Scope:
This procedure is applicable for determining protein of ground, air-dry or partially dried (90 to 95% dry matter) forage. Samples must be ground through cyclone grinder with 1 mm screen and be 90 to 95% dry matter.

Basic Principle:
Random portions of a sample are loaded into a NIR sample holder and reflected light from the ground sample is measured in the infrared region (generally 1100 to 2500 nm). Instrument is part of a system that has been calibrated using representative samples from population to be tested. Equations selected based on calibration statistics, which have been validated, are used to calculate crude protein content of feed and forage samples.

Equipment:
Near infrared reflectance spectrophotometer, wavelength range at least 1100 to 2500 nm Sample holders with infrared transmitting quartz window Computer with software for collecting, storing and processing spectra

Reagents:
None.

Safety Precautions:

• Follow manufacturer's recommendation for safe operation of instrument.

Procedure:

1. Prepare samples by the same method as the calibration samples were prepared.
2. For best results run instrument (but not lamp) continuously. If instrument is cold, warm-up time should be 1 hr.
3. Clean sample holder with a camel hair brush or vacuum. Additional cleaning may be done with soft tissue or lint-free cloth. Glass should be free of finger prints and foreign material.
4. Load NIR sample cup placing one scoop of forage (previously dried to 90-95% dry matter or greater, ground with cyclone mill to pass 1 mm screen, thoroughly mixed) on each third of the glass surface to ensure that portions of different subsamples are scanned. Overfill the sample holder and scrape off any excess.
5. Press back into holder so that sample is firmly pressed against window.
6. If any abnormality appears in window, remove back and repeat process. Consistency in sample handling, preparation, and cell packing is crucial to success.
7. Scan sample, collect spectra, and process data.

Comments:

• NIR instrument should be maintained in a dust-free environment. Clean or change filter monthly. Clean ceramic standard and drawer assembly monthly.
• Never touch or clean grating.
• NIR instrument should be maintained in a stable-temperature (25 ±3oC) room with controlled humidity (60 ±5%). Room should be free from vibration and have stable and dedicated electrical current.
• Sample must be dried and ground by the same methods as those samples used to develop the calibration equation.
• Spectrophotometer reads only a fraction of 1 mm depth of material touching glass of sample container. Therefore container must be loaded carefully with different subsample in each quadrant to make spectral reading more representative of entire sample.

Calculations:
Prediction is made by direct comparison to calibration. No additional calculations. If calibrations were made using reference values on a dry matter basis, NIR results will be expressed on a dry matter basis.

Quality Control:
Include at least one set of duplicates in each run. These duplicates must be two subsamples each packed in a separate holder. Scanning the same sample twice is not a true replicated analysis using NIR.

An acceptable average standard deviation among replicated analyses for crude protein using reference methods ranges from about ±0.10 for samples with 10% CP to ±0.20 for samples with 20% CP, which results in warning limits (2s) ranging from ±0.20 to 0.40 and control limits (3s) ranging from ±0.30 to 0.60.

Quality control for NIR analysis involves monitoring the accuracy of both the instrument and the calibration equation. Instrument diagnostics should be run and recorded weekly, after instrument warm-up, to insure that maximum response, wavelength accuracy and repeatability are within acceptable manufacturer's tolerances. For example, for NIRSystems model 6250 and 6500 instruments, maximum response should be between 55000 and 58000 (adjust if below 51000), suggested wavelength error should be less than 0.5 times currently observed error (restandardize if observed error is too large) and root mean square corrected (RMSC) repeatability of multiple scans (32 revolution and 30 scans) should be less than 20 to 30 (correct problems if greater). Each manufacturer should provide acceptable performance specifications for their instrument, and often software is provided to monitor instrument accuracy. However, it is the responsibility of the operator to run the diagnostic software routinely and record the results a minimum of once a week.

In addition to meeting specifications, instrument operation should be monitored by scanning a check sample (that has been sealed in a sample holder) each day and storing spectral data weekly. Daily results for each analysis (DM, CP, ADF, NDF, and minerals) should be plotted on an X-control chart and the chart should be examined for trends. Standard deviation (s) for the check sample can be established after 10 scans and should be substantially lower than acceptable standard deviations of duplicate reference method analyses (.10, .20, .35 and .60 for DM, CP, ADF, and NDF, respectively) because the same sample is being scanned. Results outside of ±2s upper and lower warning limits are evidence of problems with the analytical system. Results outside of ±3s upper and lower control limits are evidence of loss of control and no NIR analyses should be done without detecting and correcting the problem. Two consecutive analyses falling on one side of the mean between warning limits and control limits also indicate a loss of control.

Monitoring the calibration equation consists of two tests that determine the existence of bias and unacceptable increases in standard error of prediction corrected for bias [SEP(C)]. Every 20 to 25th sample predicted by the NIR should be analyzed by the reference method used to develop the calibration equation (Note: Any bias or increased SEP(C) can be due to inaccuracies in NIR or differences in reference method analytical procedure). A continuous time chart of observed bias and SEP(C) should be plotted to observe trends. After nine (N) samples have been accumulated, analyze these samples by the reference method (A sample size of nine is a good compromise between the number of analyses required and the statistical accuracy desired for the confidence limits given below). Calculate the observed bias and SEP(C) using the equations given below. Determine the confidence limits for bias and SEP(C) based on the standard error of the calibration equation (SEC). If the SEP(C) of the nine samples is less than the SEP(C) limit and absolute value of the bias is less than the bias limit, the calibration equation is acceptable. If the SEP(C) of the nine samples is less than the SEP(C) limit but the absolute value of the bias exceeds the bias limit, the calibration equation may be corrected by adding the bias to the intercept of the calibration equation or to each value (bias adjustment) although recalibration is recommended. If both SEP(C) and bias exceed their limits, add samples to the calibration data set and recalibrate.

NIR Quality Control Calculations:

Di = Xi - Yi
Bias = Di / N
BiasConfidence Limit = ±0.55 X (SEC)
SEP(C) Confidence Limit = 1.29 X (SEC)

• Di = difference
• Xi = reference method value
• Yi = NIR value for ith sample
• N = 9 (number of samples)
• Bias = average difference between reference and NIR values
• SEP(C) = standard error for prediction corrected for bias
• SEC = standard error of the calibration equation.

This monitoring procedure can be used with any NIR instrument and some manufacturers have incorporated this approach into their quality control monitoring software.

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