GB/T 5009.206-2007Determination of tetrodotoxin in aquatic products -- HPLC-fluorescence detection method (English PDF)

3.1 Reagent

3.1.1 glacial acetic acid (CH3COOH).

3.1.2 sodium hydroxide (NaOH).

3.2 Reagent preparation

3.2.1 acetic acid (0.5%). 5mL of glacial acetic acid diluted with water to 1L.

3.2.2 sodium hydroxide solution (1mol/L). 40g of sodium hydroxide dissolved in water and dilute to 1000mL.

3.3 Standard

Tetrodotoxin (C11H17N3O8, CAS number. 4368-28-9), purity >=98%, or certified by the state and granted a certificate of standard reference materials Reference material.

Boiling water bath for 10min, with constant stirring to prevent clumping tissue. Cooled to room temperature, 20000g high-speed centrifugal 20min, supernatant was pipetted 25mL volumetric flask. Acetic acid solution was added to the precipitate (0.5%) 11mL, mix thoroughly, boiled in a boiling water bath for 5min, stirring constantly to prevent Only organization agglomeration. Cooled to room temperature, 20000g high-speed centrifugal 20min, the supernatant was pipetted. The combined supernatant was mixed with a solution of acetic acid (0.5%) volume to 25mL. This extract is equivalent to 0.4g 1mL sample.

5.2.2 Liver tissue samples

Accurately weighed 10g (to the nearest 0.1g) in 50mL centrifuge sample tube, was added acetic acid (0.5%) 8mL, mix well, at room temperature High speed centrifugation at 20000g 20min, supernatant was pipetted 25mL volumetric flask. 8mL extracted repeatedly with acetic acid (0.5%) 1 time. The combined supernatant was mixed with a solution of acetic acid (0.5%) volume to 25mL. This extract is equivalent to 0.4g 1mL sample.

5.2.3 Skin tissue sample

Accurately weighed 10g (to the nearest 0.1g) in 50mL centrifuge sample tube, was added acetic acid (0.5%) 11mL, mix well, chamber High speed centrifugation at 20000g temperature 20min, supernatant was pipetted 25mL volumetric flask. The extraction was repeated with 11mL acetic acid (0.5%) 1 time. The supernatant was combined with a solution of acetic acid (0.5%) volume to 25mL. This extract is equivalent to 0.4g 1mL sample.

Note 1. The samples were prepared, frozen samples need to be operated in a semi-thawed state. If not timely test sample, should be stored at 4 °C, tested within 24h.

Note 2. If the case where the puffer fish organs or tissue sample of less than 10g, is extracted according to the above method of operation, need to reduce the amount of acetic acid (0.5%) added.

Note 3. extract should contain not be separated from the liquid surface of the oil was centrifuged, gum or lipids.

5.3 Mouse assay

Select 19.0g ~ 21.0g of specific pathogen free (SPF) male Kunming 6 healthy mice were weighed and body weight was recorded. Randomly divided of the experimental group and the control group two, three per group. Extract or acetic acid solution to each test mouse by intraperitoneal injection of 1mL sample (0.5%) (control solution). If overflow injection during injection, the mice should be discarded and a re-injected mice. Record Note Shooting start and finish time, carefully observe and record the time of death when the mice stopped breathing (to the last breath is only a mouse).

If the sample extract after injection, mice were less than the time of death 7min, press Appendix A Table A.1 calculated 1mL sample extract Virulence, as a reference, formulated ambassador mice died 7min ~ 13min required dilution, use solution of acetic acid (0.5%) in dilute Release, the diluent re-injected mice at least 3 to determine the virulence of the sample. Specific examples in Appendix B. If the sample solution injection, mice Time of death is greater than or equal to 7min, virulence is determined directly from the sample median lethal time.

Symptoms of poisoning death in mice. Mice were injected with tetrodotoxin quiet early, followed by difficulty in breathing, shortness of breath, abdominal contraction accelerated, Unresponsive, then sudden Rush, sprint jerks, flee, moved, flipped jumping operation like struggling, after several tens of seconds, mice Leg twitch violently, after 2 or 3 times to climb lying immobile, weak abdominal breathing gradually slowed down, and finally death. To stop breathing as a judgment of death standard.

6.1 Calculate virulence

The time to death of mice is obtained in the test, the median lethal time is calculated, and the calculated Table A.1 virulence (if the bit when the time is right in the middle of the number of lethal two times given in Table A.1, whichever is greater of the two values of the time period; if given in Table A.1 of between when the time out of the two, but not exactly in the middle, then take a time value closer to the median time to death). If the mouse body weight is not exactly 20.0 g of, as shown in Table A.2 murine unit (MU) for correcting the extraction is shown by the mouse virulence was corrected unit.

Is defined such that 1MU weighing 20.0g of specific pathogen free (SPF) male Kunming mice virulence 30min death phase when to 0.18µg tetrodotoxin.

Tetrodotoxin content in the samples according to the formula (1).

X = M × E × f × W (1) where.

X-tetrodotoxin content --- sample, in units of units per gram rats (MU/g);

Rat number M --- 1mL injection unit, in units of murine units (a MU);

E --- extraction coefficient, the method for the extraction coefficient 3.11;

f --- Sample dilution liquid extract;

--- W is the weight correction coefficient mice.

6.2 Report the results

In the control group of normal mice, the puffer fish tetrodotoxin content in various tissues is determined and expressed as follows.

If the time of death in mice is greater than 30min, results are reported as 7 Other the detection limit of this method is 3.11MU/g.

The second method of liquid chromatography - tandem mass spectrometry Principle 8 Sample with 1% acetic acid - methanol solution was extracted, purified immunoaffinity column, liquid chromatography - tandem mass spectrometry, external standard.

9.1 Reagent

9.1.1 methanol (CH3OH). chromatography.

9.1.2 acetonitrile (CH3CN). HPLC grade.

9.1.3 formic acid (HCOOH). chromatography.

9.1.4 glacial acetic acid (CH3COOH). excellent pure.

9.1.5 ammonium acetate (CH3COONH4). chromatography.

9.1.6 dodecahydrate disodium hydrogen phosphate (Na2HPO4 · 12H2O).

9.1.7 sodium phosphate dihydrate (NaH2PO4 · 2H2O) dihydro.

9.1.8 Sodium chloride (NaCl).

9.1.9 sodium hydroxide (NaOH).

9.2 Reagent preparation

9.2.1 acetate - methanol (199). The glacial acetic acid and methanol at a volume ratio of 199 were mixed to homogeneity.

9.2.2 acetate - methanol (298). The glacial acetic acid and methanol at a volume ratio of 98 2 were mixed to homogeneity.

9.2.3 formic acid (1999). 1mL of formic acid was added to the 999mL of water, and mix.

9.2.4 0.1% formic acid solution containing 5mmol/L ammonium acetate. Weigh 0.19g of ammonium acetate, 0.1% formic acid solution was brought to 500mL.

9.2.5 formic acid (0.1%) - acetonitrile (11). formic acid (0.1%) mixed with an equal volume of acetonitrile.

9.2.6 phosphate buffer solution (PBS solution). Weigh-hydrogen phosphate dodecahydrate, disodium 6.45g, sodium dihydrogen phosphate dihydrate 1.09g, chloride Sodium 4.25g, dissolved in water and dilute to 500mL.

9.2.7 sodium hydroxide solution (1mol/L). 20.0g of sodium hydroxide was weighed accurately, dissolved in water and dilute to 500mL.

9.3 Standard

Tetrodotoxin (C11H17N3O8, CAS number. 4368-28-9), purity >=98%, or certified by the state and granted a certificate of standard reference materials Reference material.

9.4 Standard Preparation

9.4.1 tetrodotoxin Standard stock solution (100µg/mL). Weigh accurately 10 mg tetrodotoxin standard (accurate to 0.1mg), with a small amount of methyl Acid solution (0.1%) was dissolved to methanol to 100mL, -18 °C below stored.

9.4.2 tetrodotoxin intermediate standard solution (1µg/mL). exact amount of tetrodotoxin standard stock solution (100µg/mL) amount to formic acid (0.1%) - was diluted in acetonitrile (1 1) and set the volume to prepare a standard intermediate liquid 1µg/mL, here -18 °C stored.

9.4.3 tetrodotoxin series of standard working solution. accurately draw tetrodotoxin intermediate standard solution (1µg/mL) amount to formic acid (0.1%) -Acetonitrile (11) and set the volume was diluted to give a concentration of 1µg/L ~ 1000µg/L of the working standard solution series, using now.

9.5 Material

9.5.1 tetrodotoxin immunoaffinity column. dimensions 3mL, the maximum capacity of the column 1000ng, or equivalent column.

The organic phase 9.5.2 0.22µm microporous membrane.

11.1 Preparation of samples

After washing with water the surface of the fish waste, fish surface moisture absorbed by filter paper with scissors decomposed into the fish muscle, liver, skin, and of Glands (testis or ovaries) and other parts, each part of the tissue with blood wash with water, dry paper tissue after water on the surface of each cut into pieces, were sufficiently Mass, into a clean container, and labeled tag.

11.2 Sample collection

5g weighed (accurate to 0.01g) sample was homogenized 50mL stoppered centrifuge tube, was added 11mL acetic acid - methanol solution (199), vortex Rotary oscillation 2min, 50 °C water bath ultrasonic extraction 15min, at 8000r/min centrifugal 5min, supernatant was transferred to a 25mL volumetric flask.

The residue was added 11mL acetic acid - methanol solution (199), the extraction was repeated once, the supernatant was combined with acetic acid - methanol (199) fixed Volume to 25mL. The extract was pipetted to the other about 10mL 50mL stoppered centrifuge tube, -20 °C frozen 30min, then at 8000r/min from Heart 5min, 5mL accurate pipetting the supernatant was 20mLPBS solution is diluted with sodium hydroxide solution (1mol/L) adjusted at pH Range of 7 to 8, to be purified.

Purification Sample 11.3 the immunoaffinity column in PBS natural sequestration discharge flow rate, liquid to be purified into a flow rate of 1 drop/s and passed through the column, then 10mL water rinsed, drained, with 5mL of acetic acid - methanol solution (298), eluent dry nitrogen at 45 °C, formic acid was added 1.0mL Solution (0.1%) - acetonitrile (11) was dissolved the residue, ultrasonic 1min, after the organic phase through a microporous membrane 0.22µm for liquid chromatography - Cascade with mass spectrometry.

11.4 Instrument reference conditions

HPLC conditions refer to 11.4.1 HPLC conditions are listed below with reference to.

a) Column. GelAmide-80 column, length 150mm, inner diameter 2mm, particle size 5 m, column or equivalent.

b) flow rate. 0.3mL/min.

c) Column temperature. 40 °C.

d) Injection volume. 10µL.

e) The mobile phase and gradient conditions are shown in Table 1. Mobile phase Solution A. 0.1% formic acid solution containing 5mmol/L ammonium acetate; mobile phase Solution B. acetonitrile.

Table 1 Mobile phase and gradient conditions

Time/min A /% B /%

0.0 1090

2.0 10 90

2.1 90 10

6.0 90 10

6.1 1090

8.0 1090

11.4.2 Mass Reference Conditions

Mass spectrum Reference conditions are listed below.

a) ion source. electrospray ionization source (ESI);

b) scan mode. positive ion scan;

The standard series tetrodotoxin working solution were injected into the liquid chromatography - tandem mass spectrometer, the measured peak area corresponding to the standard series of work Concentration of the solution as the abscissa tetrodotoxin, TTX peak area of peaks of the vertical axis, the standard curve.

11.6 Determination of the sample solution

The sample solution is injected into the liquid chromatography - tandem mass spectrometer, the peak area obtained in response, the test solution to give a standard curve tetrodotoxin the concentration of pigment. Tetrodotoxin extracted ion chromatograms of standard solutions See Appendix C in FIG C.1.

11.7 Qualitative

Under the same test conditions, the sample solution is compared to the retention time of the standard solution of tetrodotoxin, a deviation within ± 2.5%, and the subject Measured the relative abundance of ions, it should be consistent with the concentration of the standard working solution similar relative abundances of ions, which should be consistent with the abundance ratio deviation Table 3 Claim.

Table 3 Maximum allowable deviation relative ion abundances

The relative ion abundances > 50% > 20% ~ <=50% > 10% ~ <=20% <=10% Permissible relative deviation of ± 20% ± 25% ± 30% ± 50%

11.8 Blank test

Except that no sample addition are by the measuring step.

16.2 Reagent preparation

16.2.1 Sodium hydroxide solution (1mol/L). Weigh 40g of sodium hydroxide, dissolved in water and diluted to 1000mL.

16.2.2 phosphate buffered saline (PBS solution). Weigh 6.45g of disodium hydrogen phosphate dodecahydrate, 1.09 g of sodium dihydrogen phosphate dihydrate, 4.25g of sodium chloride, dissolved in water and dilute to 500mL.

16.2.3 acetate - methanol (199). The glacial acetic acid and methanol at a volume ratio of 199 were mixed to homogeneity.

16.2.4 acetate - methanol (298). The glacial acetic acid and methanol at a volume ratio of 98 2 were mixed to homogeneity.

16.2.5 ammonium acetate buffer solution. Weigh 4.6g of ammonium acetate and 2.02g of heptane sulfonate, was dissolved by adding about 700mL of water, adjusted with glacial acetic acid pH 5.0, diluted with water to 1L.

16.2.6 sodium hydroxide solution (4mol/L). Weigh 160g of sodium hydroxide, dissolved in and diluted to 1L with water.

16.2.7 acetic acid solution (1%). glacial acetic acid and water at a volume ratio of 199 were mixed to homogeneity.

16.3 Standards

Tetrodotoxin (C11H17N3O8, CAS number. 4368-28-9), purity >=98%, or certified by the state and granted a certificate of standard reference materials Reference material.

16.4 Standard Preparation

16.4.1 standard stock solution (100µg/mL). Weigh accurately 10 mg tetrodotoxin standard (accurate to 0.1 mg), was dissolved with a small amount of water.

In methanol to 100mL. It can be stored at 4 °C month.

16.4.2 standard working solution. stock standard solution appropriate amount according to need, standard working solution diluted to an appropriate concentration in a solution of acetic acid (1%).

Pro with the existing service.

16.4.3 matrix-matched series of standard working solution. Prepare a standard solution in a series of blank substrate working solution of appropriate concentration. Using now.

16.5 Materials

16.5.1 tetrodotoxin immunoaffinity column. dimensions 3mL, the maximum capacity of the column 1000ng, or equivalent column.

16.5.2 0.22µm aqueous phase microporous membrane.

18.1 Preparation of samples

After washing with water the surface of the fish waste, fish surface moisture absorbed by filter paper with scissors decomposed into the fish muscle, liver, skin, and of Glands (testis or ovaries) and other parts, each part of the tissue with blood wash with water, dry paper tissue after water on the surface of each cut into pieces, were sufficiently Mass, into a clean container, and labeled tag.

Sample Extraction 18.2 2g were weighed (accurate to 0.01g) sample was placed in a 50mL polypropylene centrifuge tube, was added 10mL acetic acid - methanol solution (199), Zhen Swing 2min, 60 °C water bath ultrasonic extraction 30min, 4500r/min centrifugal 5min. 60 deg.] C the supernatant was concentrated by rotary evaporation to near dryness, Into 2mL acetic acid solution (1%) to fully dissolve the residue, transferred to a 10mL polypropylene centrifuge tube, was added 8mLPBS diluted with hydrogen Sodium hydroxide solution (1mol/L) pH was adjusted to 7-8, to be purified.

Purification Sample 18.3 After the immunoaffinity column warm to room temperature, remove the plug affinity column, the column storage solution discharged, the liquid to be purified into 10mL. Full cleaning fluid After the outflow, rinsed with 10mL water, a suitable fluid pressure within the column squeezed. With 4mL acetic acid - (298) eluting with methanol, wash the collected Deliquoring dry nitrogen at 50 deg.] C, dissolved in acetic acid solution (1%) and dilute to 1mL, by the water phase 0.22µm filter membrane, the filtrate for Liquid Chromatography - Fluorescence Analysis.

18.4 Instrument reference conditions

HPLC conditions refer to 18.4.1 HPLC conditions are listed below with reference to.

a) Column. C18 column, length 250mm, internal diameter 4.6mm, particle size 5 m, column or equivalent.

b) mobile phase. acetonitrile - ammonium acetate buffer (595, volume ratio).

c) flow rate. 1.0mL/min.

d) Column temperature. 30 °C.

e) Detection wavelength. excitation wavelength 385nm, emission wavelength 505nm.

f) Injection volume. 40µL.

Post-column derivatization 18.4.2 reference condition Post-column derivatization with reference conditions are listed below.

a) a solution derived from. sodium hydroxide (4mol/L).

b) derived solution flow rate. 0.5mL/min.

c) derived tube temperature. 110 °C.

18.5 Standard curve prepared

The matrix-matched series of standard working solution were injected into the liquid chromatography, measuring the peak area corresponding to the working standard solution of tetrodotoxin the concentration of abscissa, tetrodotoxin peak area of peaks of the vertical axis, the standard curve. HP...