Standardization and determination of marker compounds characterizing mature silkworm (Bombyx mori) powder

Figures & data

Figure 3. Calibration curves and residual distribution plots of serine, glycine, and alanine. (a), (b), and (c) show calibration curve of serine, glycine, and alanine, respectively. (d), (e), and (f) show residual distribution plot of serine, glycine, and alanine, respectively.

Figure 1. Manufacturing process of mature silkworm powder.

Table 1. HPLC operation conditions for amino acid analysis.

Instrument	Shiseido Nanospace SI-2
Column	Capcell Pak C18 MG (3.0 mm × 150 mm, 3 μm)
Mobile Phase	A: 35 mM CH3COONa, B: 90% Acetonitrile
Detector	UVD (436 nm)
Elution System (Gradient)	Time	A %	B %
	0	82	18
	5	82	18
	35	55	45
	37	40	60
	45	40	60
	45.1	82	18
	55	82	18
Injection volume	5 μL
Column Temperature	40°C.
Run time	55 min
Flow rate	0.4 mL/min

Figure 2. HPLC chromatograms of serine, glycine, and alanine. (a) Chromatogram of the amino acids in the standard mixture, (b) Chromatogram of the amino acids in processed mature silkworm powder.

Table 2. Precision and accuracy of serine, glycine, and alanine analysis in processed mature silkworm powder.

Standard		Concentration (μg/mL)	Mean ± SD (μg/mL)	Relative standard deviation (%)	Recovery (%)
Serine	Intra-day	100	98.82 ± 0.36	1.73	98.82
		25	24.84 ± 0.11	1.18	99.37
		6.25	6.19 ± 0.08	0.77	99.04
	Inter-day	100	102.34 ± 534	5.63	102.34
		25	24.82 ± 0.26	2.03	99.3
		6.25	5.99 ± 0.19	2.89	95.85
Glycine	Intra-day	100	100.03 ± 0.57	1.90	100.03
		25	26.25 ± 0.22	0.75	105.00
		6.25	6.25 ± 0.39	2.49	98.42
	Inter-day	100	100.43 ± 1.13	3.51	104.43
		25	26.19 ± 0.25	4.57	104.76
		6.25	6.02 ± 0.24	4.39	96.32
Alanine	Intra-day	100	99.38 ± 0.30	1.39	99.38
		25	25.98 ± 0.23	0.55	103.91
		6.25	5.68 ± 0.19	0.97	90.94
	Inter-day	100	102.50 ± 4.55	4.61	102.5
		25	26.07 ± 0.61	2.00	104.27
		6.25	5.82 ± 0.19	3.15	93.05

Table 3. Limit of quantification (LOQ) and limit of detection (LOD) of serine, glycine, and alanine.

	LOQ (μg/mL)	LOD (μg/mL)
Serine	0.88	0.33
Glycine	3.37	1.11
Alanine	2.79	0.92

Table 4. Effect of producing area and season on the contents of serine, glycine, and alanine in processed mature silkworm powder.

Area	Sancheong Kyungnam		Boeun Chungnam		Buan Jeounbuk		Mixture of three batches from different areas produced in spring
Season	Spring	Fall	Spring	Fall	Spring	Fall	Spring
Serine (mg/g)	69.00 ± 1.57*	-	55.28 ± 4.46*	57.32 ± 3.06*	61.31 ± 0.22*	58.39 ± 1.16*	61.08 ± 3.27*
Glycine (mg/g)	118.96 ± 9.04*	-	92.01 ± 8.73*	98.25 ± 5.14*	109.28 ± 8.32*	101.02 ± 2.07*	102.30 ± 7.49*
Alanine (mg/g)	96.91 ± 6.35*	-	75.49 ± 7.85*	81.17 ± 4.34*	89.45 ± 6.88*	84.16 ± 1.80*	82.34 ± 7.33*
Sum (mg/g)	284.87 ± 16.73*	-	222.78 ± 20.62*	236.74 ± 12.54*	260.03 ± 15.36*	243.57 ± 5.02*	245.73 ± 18.05*
Threshold of marker compounds suggested		Sum of the three amino acids: serine, glycine, and alanine, 200–300 mg/g

*Each value represents the mean± SD (n = 3).