Sub-picogram determination of Vitamin B12 in pharmaceuticals and human serum using flow injection with chemiluminescence detection

Analytica Chimica Acta 488 (2003) 71–79

Sub-picogram determination of Vitamin B12 in pharmaceuticals and human serum using flow injection with chemiluminescence detection Zhenghua Song∗ , Shuang Hou Department of Chemistry, Northwest University, Xi’an 710069, PR China Received 20 January 2003; received in revised form 23 May 2003; accepted 23 May 2003

Abstract A sensitive chemiluminescence (CL) method, based on the enhancive effect of cobalt(II) on the CL reaction between luminol and dissolved oxygen in a flow injection (FI) system, was proposed for determination of Vitamin B12 . The increment of the CL intensity was proportional to the concentration of Vitamin B12 , giving a calibration graph linear over the concentration from 2.0 ×10−10 to 1.2 ×10−6 g l−1 (r 2 = 0.9992) with the detection limit of 5.0 ×10−11 g l−1 (3σ). At a flow rate of 2.0 ml min−1 , a complete determination of Vitamin B12 , including sampling and washing, could be accomplished in 0.5 min with the relative standard deviations (R.S.D.) of less than 5.0%. The proposed method was applied successfully to the determination of Vitamin B12 in pharmaceuticals, human serum, egg yolk and fish tissue. © 2003 Elsevier B.V. All rights reserved. Keywords: Vitamin B12 ; Chemiluminescence; Flow injection; Pharmaceuticals; Human serum; Egg yolk; Fish tissue

1. Introduction Vitamin B12 , also known as cobalamin (C63 H88 CoN14 O14 P) containing a cobalt ion within tetrapyrrole ring, is a very unusual biochemical species. Vitamin B12 is naturally found in animal foods including fish, milk or milk products, eggs, meat, and poultry [1]. It helps maintain healthy nerve cell and red blood cell, and is also necessary to make DNA, the genetic material in all cells [2,3]. The deficiency of Vitamin B12 may lead to fatigue, weakness, nausea, constipation, weight loss, and even as severe as addisonian pernicious anemia [4]. Recently, considerable interests have been focused on Vitamin B12 , not only for its application in nutrition, but also for the ∗ Corresponding author. Fax: +86-29-8303511. E-mail address: [email protected] (Z. Song).

potential therapeutic effect on shaky-leg syndrome [5], atherosclerosis [6] and heart disease [7]. Vitamin B12 is most often determined by the several methods, generally based on the determination of cobalt(II) contained in Vitamin B12 , including spectrophotometry [8–10], AAS [11] and voltammetry [12,13]. In addition, other analytical techniques have also been applied in the determination of Vitamin B12 in complicated matrices, involving biological methods such as radioassay and optical sensor [14,15], HPLC [16–19], capillary electrophoresis [20] and fluorescence spectrometry [21,22]. Chemiluminescence (CL) is a simple, sensitive and selective analytical method, offering an attractive alternative for determination of both organic and inorganic species [23,24]. In addition, two CL methods, on the basis of luminol–hydrogen peroxide–cobalt(II) CL system, have been reported for determination of Vitamin B12

0003-2670/03/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0003-2670(03)00665-2

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in tablets with the detection limits of 2.0 × 10−5 [25] and 3.5 × 10−7 g l−1 [26], respectively. In this paper, it was found that the cobalt(II), liberated from Vitamin B12 , could greatly enhance the CL intensity between luminol and dissolved oxygen reaction. Under the optimized conditions, the increment of the CL intensity was linear over the Vitamin B12 concentration from 2.0 × 10−10 to 1.2 × 10−6 g l−1 with a detection limit of 5.0 × 10−11 g l−1 Vitamin B12 (3σ) and the relative standard deviations (R.S.D.) of less than 5.0%. Moreover, at a flow rate of 2.0 ml min−1 , a whole determination of Vitamin B12 , including sampling and washing, could be completed in 0.5 min, offering a throughput of 120 h−1 accordingly.

2. Experimental 2.1. Reagents Vitamin B12 was supplied by the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Luminol (Fluka, Biochemika) was obtained from Xi’an Medicine Purchasing and Supply Station, China. Cobalt was spectrographically standardized substance (Johnson Matthey & Co. Ltd.). All other chemicals used were of analytical-reagent grade. Water, purified in a Milli-Q system (Millipore, Bedford, MA, USA), was used throughout the experiments.

A stock solution of Vitamin B12 (1.0 g l−1 ) was prepared by dissolving 0.100 g of crystalline Vitamin B12 with 0.1 mol l−1 hydrochloric acid to 100 ml in a brown calibrated flask. A standard solution for calibration was prepared freshly from the stock solution after acidification before analysis. Luminol was used as supplied to prepare a 0.25 mol l−1 stock standard solution in 1000 ml of 0.5 mol l−1 sodium hydroxide in a calibrated flask. A standard solution of cobalt(II) (1.0 g l−1 ) was prepared as stock solution. All samples and standard solutions were prepared and stored in the brown calibrated flask at 4 ◦ C. 2.2. Apparatus The FI system was shown schematically in Fig. 1. The manifold included a peristaltic pump (Shanghai Meter Electromotor Plant, Model ND-15, 15 rpm) for pumping each of all flow streams at a flow rate of 2.0 ml min−1 . PTFE tubing (1.0 mm i.d.) was used to connect all components in the FI system. And a 100 ␮l loop of six-way valve was used to inject Vitamin B12 quantitatively into carrier stream. A mixing tube of 2.0 cm was applied for mixing the Vitamin B12 , luminol and sodium hydroxide before the solutions approached the flow CL cell. The CL flow cell is a flat spiral consisting glass tubing (2.0 mm i.d., 15.0 cm length) placed adjacent to PMT. The CL intensity generated in the CL flow cell was detected without wavelength discrimination with the negative

Pump

NaOH

CL Flow Cell

Sample

Detector Mixing

Carrier

Tubing

Recorder

Valve Luminol Waste Fig. 1. Schematic diagram of the flow-injection system for determination of Vitamin B12 sodium hydroxide: 3 × 10−2 mol l−1 ; luminol: 1.0 × 10−5 mol l−1 ; flow rate: 2.0 ml min−1 ; high voltage: −750.

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high voltage of 750 V supplied to the PMT by a luminosity meter (Xi’an Keri Electron Device Ltd., Model GD-1), which is connected with a recorder (Shanghai Dahua Instrument and Meter Plant, Model XWT-206). 2.3. Procedures The carrier water and the solutions (including sodium hydroxide, luminol and sample solutions) were propelled at the constant flow rate on each flow lines. Until a stable baseline was recorded, 100 ␮l of Vitamin B12 was injected into carrier stream by six-way valve quantitatively, and then the solution was merged with the luminol stream. Then the mixing solution was delivered into the CL flow cell in an alkaline medium to generate the CL, which was thereafter detected by luminometer. Thereby the concentration of Vitamin B12 could be determined by measuring the increase of the CL intensity, I = Is − I0 , where I0 and Is are CL signals in the absence and in the presence of Vitamin B12 , respectively. 2.4. Determination of Vitamin B12 in pharmaceutical preparations An amount of 0.1 mol l−1 hydrochloric acid was employed to acidify pharmaceuticals according to the literature [25]. One milliliter of the Vitamin B12 injection, containing 0.5 mg Vitamin B12 , was acidified by 10 ml of hydrochloric acid, and then the solution was diluted into 25 ml. As to Vitamin B12 tablets, not less than 20 tablets were weighed and ground to fine powder. And then a sample containing approximate 500 ␮g of Vitamin B12 was weighed accurately, transferred into a 100 ml brown calibrated flask and made up to volume with water. Then, a portion of 0.5 ml prepared sample was transferred to a 25 ml brown calibrated flask containing 5.0 ml of hydrochloric acid and make up to the volume with water. As the procedure proposed, the pharmaceutical preparation samples were determined directly after being diluted appropriately. The Pharmacopoeia method [27] was applied as reference method, and according to the procedure recommended, pharmaceutical preparations were made approximate 2.5 × 10−2 g l−1 solutions which were then determined at 361 nm by UV.

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2.5. Determination of Vitamin B12 in human serum The samples of human serum were supplied by Radio-Immunity Center, People’s Hospital of Shannxi Province. The analytical procedure was carried out as follows: 0.1 ml of each sample was acidified with 1.0 ml of 0.1 mol l−1 hydrochloric acid, and then diluted in to 10 ml. And after 10-fold dilution, the samples were determined by proposed method directly. 2.6. Determination of Vitamin B12 in egg yolk and fish tissue The eggs were purchased from the local market and the dried fish tissue was supplied by Fisheries Research Institute of Shannxi. Both kinds of samples were pretreated according to the literature [28]. Approximate 5.0 g of boiled hen egg yolk was weighed, grounded and acidified with 50 ml hydrochloric acid (0.5 mol l−1 ) in digester. Each of hen egg yolk sample has been digested ultrasonically until homogeneous mixture is obtained; the supernatant solution of centrifuged sample (4 × 103 rpm) was then filtrated and the filtrate was determined directly by the proposed CL method after appropriate dilution. The dried muscle (1.0 g) or liver (0.2 g) tissue of fish were ground to powder, and then mixed with 10 ml hydrochloric acid of 0.5 mol l−1 . The solution was homogenized ultrasonically and the digested sample was centrifuged, and the supernatant solution was filtrated. The resulting sample was determined by the presented method directly after dilution. 2.7. Determination of Vitamin B12 in spiked human serum and urine samples The serum samples supplied by the Hospital of Northwest University and the urine samples collected from three volunteers were spiked prior to determination. 75.0 or 50.0 ␮g Vitamin B12 for serum and 10.0, 7.5 or 5.0 ␮g for urine were spiked into 1.0 ml of samples, respectively. After homogenization, 0.1 ml aliquot of the spiked sample was acidified with 1.0 ml of hydrochloric acid and then diluted to 50 ml. After dilution with a factor of 1.0 × 104 for serum samples and 1.0 × 102 for urine samples, the samples were determined by the proposed method directly.

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3. Results and discussion

evident that carbonate inhibited the luminol–dissolved oxygen–Vitamin B12 CL signal. As illustrated in Fig. 2 (curve III), the relative CL intensity in the presence of carbonate decreased from 622 down to 332 by 46.6%.

3.1. Time profile of the CL reaction Before carrying out the FI method, the kinetic curve was examined by static method. The time profile of the reaction of luminol-dissolved oxygen-acidified Vitamin B12 system was tested using 1.0 × 10−5 mol l−1 luminol in 0.02 mol l−1 sodium hydroxide solution. It was found that the rate of the CL reaction in solution was comparatively slow in the absence of acidified Vitamin B12 . And as shown in Fig. 2 (curve I), CL intensity reached the maximum at 5 s after the reactants were mixed, and then became extinguished within 80 s thereafter. Also, it can be seen that a scintillescent CL signal was detected in the presence of acidified Vitamin B12 (1.0 × 10−5 g l−1 ), and the CL intensity reached maximum at 1.5 s, then tended to be vanishing in following 70 s (curve II), giving a maximum intensity 41-fold as that in the absence of Vitamin B12 . Considering the enhancive effect of carbonate in some CL systems [29–31], the time profile of luminol–dissolved oxygen–Vitamin B12 –carbonate CL system was also tested at the same condition. It was

3.2. Effect of luminol concentration The influence of luminol concentration on CL was examined. Through the determining a series of standard solutions of Vitamin B12 (2.0 × 10−9 to 2.0 × 10−7 g l−1 ) by using different concentrations of luminol solution from 1.0 × 10−7 to 1.0 × 10−4 mol l−1 , it was found that luminol concentrations of 1.0 × 10−5 mol l−1 offered the higher sensitivity than any other concentrations of luminol. Therefore, 1.0 × 10−5 mol l−1 luminol was chosen for the subsequent experiment. 3.3. Effect of sodium hydroxide concentration Owing to the nature of luminol CL reactions, which is more favored in alkaline medium, sodium hydroxide was introduced into the CL cell through a flow line to improve the sensitivity of the system. A series

700 Relative CL intensity

II

600 500 400

III

300 200 100

I

0 0

2

4

6

8

10

12

14

16

18

20

Time (s) Fig. 2. Kinetic CL–rime profile in static system. (I) CL intensity of luminol–dissolved oxygen. (II) CL intensity of luminol–dissolved oxygen–acidified Vitamin B12 . (III) CL intensity of luminol–dissolved oxygen–acidified Vitamin B12 –CO3 2− luminol: 1.0 × 10−5 mol l−1 ; Vitamin B12 : 1.0 × 10−5 g l−1 ; carbonate: 1.0 × 10−5 g l−1 ; sodium hydroxide: 2.0 × 10−2 mol l−1 ; high voltage: 750 V.

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of sodium hydroxide solutions from 5 × 10−3 to 0.1 mol l−1 were tested in the presence of 5.0 × 10−8 g l−1 Vitamin B12 and 1.0 × 10−5 mol l−1 luminol. The CL intensity (I) versus sodium hydroxide concentration plot reached a maximum at about 3.0 × 10−2 mol l−1 sodium hydroxide, thus this concentration was employed in subsequent experiments. 3.4. Influence of sample pH on CL The influence of sample pH on CL was investigated by examining the CL intensity in the presence of 5.0× 10−8 g l−1 Vitamin B12 with the different pH values from 1.2 to 6.0. It was found that the CL intensity (I) increased steeply with the pH values varying from 1.2 to 3.0, and then tended to be stable with the pH values from 3.0 to 6.0. So the sample pH was adjusted around 4.5 for the following experiments. 3.5. Effectiveness of acidification of Vitamin B12 It is very important to evaluate the effectiveness of acidification of Vitamin B12 to liberate cobalt, which was highly dependent on hydrochloric acid concentration. In the experiment, 1.25 ␮g Vitamin B12 was acidified with 10 ml hydrochloric acid from 0.05 to 0.5 mol l−1 , respectively. And then, the solutions containing 5.0 × 10−8 g l−1 Vitamin B12 were determined by the proposed method. The effectiveness of acidification was characterized by recovery, which varied from 99.34 to 101.37% with the hydrochloric acid concentrations more than 0.1 mol l−1 . Thus, 0.1 mol l−1 hydrochloric acid was employed to acidify Vitamin B12 samples in the experiments. 3.6. Effect of the length of mixing tubing and flow rate The mixing tubes from 0.5 to 6.0 cm were employed respectively to test the effect of the length of mixing tube on CL intensity. By comparing the CL intensities by using different length of mixing tube in the presence of 5.0 × 10−7 g l−1 Vitamin B12 , it could be observed that the CL intensity was much stronger using 1.0 cm mixing tube than that using any other mixing tube. Accordingly, 1.0 cm was then selected as the optimum length of mixing tube.

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The influence of flow rate on determination was examined by investigating the signal-to-noise ratio under different flow rates. And the flow rate of 2.0 ml min−1 offering the highest signal-to-noise ratio was then chosen as suitable condition considering analytical precision. 3.7. Performance of proposed method for Vitamin B12 determination A series of standard solutions of Vitamin B12 were injected into the manifold depicted in Fig. 1. The increment of the CL intensity was found to be proportional with the concentration of acidified Vitamin B12 , and the calibration graph was linear from 2.0 × 10−10 to 1.2 × 10−6 g l−1 with the detection limit of 5.0 × 10−11 g l−1 . And the regression equation is I = 1.48CVitamin B12 + 3.42

and

r 2 = 0.9992.

The R.S.D. of five determinations were 4.53, 3.79, 2.03 and 1.56% with Vitamin B12 concentration of 7.0 × 10−10 , 7.0 × 10−9 , 7.0 × 10−8 and 7.0 × 10−7 g l−1 , respectively. At a flow rate of 2.0 ml min−1 , a complete determination of analyte, including sampling and washing, could be accomplished in 0.5 min, giving a throughput of 120 h−1 with a R.S.D. of less than 5.0%. 3.8. Interference studies The interference of foreign substances were tested by analyzing a standard solution of Vitamin B12 (5.0× 10−9 g l−1 ) into which increasing amounts of interfering analyte was added. The tolerable ratios of foreign species with respect to 5.0 × 10−9 g l−1 Vitamin B12 for interference at 5.0% level were more than 1.0 × 106 for Cl− , NO3 − , Ac− , I− , SO4 2− , PO4 3− , BrO3 − , Na+ , amylum, glucose, borate, oxalate, and malic acid, 5×105 for NH4 + , Mg2+ , Ca2+ , methanol, ethanol, glutin, Tween-80, and CTMAB, 1.0 × 105 for Ba2+ , Pb2+ , urea, uric acid, and tartrate, 2.0 × 104 for Cu2+ , Zn2+ , Ni2+ , Cr3+ , and Fe2+ /Fe3+ , 1.0 × 104 for VC, 4.0 × 103 for Vitamin B1 and Vitamin B2 , and 2.0 × 102 for carbonate and bicarbonate, respectively. Ligands including CN− , SCN− , 8-hydroxyquinoline and phenanthroline reduce the CL seriously with the tolerable ratio as low as 2.

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3.9. Applications 3.9.1. Determination of the Vitamin B12 in pharmaceutical preparations Following the method described under Section 2.4, the determination for Vitamin B12 was carried out on the pharmaceutical preparations including injections and the tablets, which were purchased from the local market. Vitamin B12 in pharmaceutical injections and tablets were determined by standard addition method, into which a known quantity of Vitamin B12 was added. The results were listed in Table 1 with the recoveries varying from 97.6 to 107.3% and R.S.D. of less than 3%. To verify the results obtained by the proposed method, reference method recommended by The Pharmacopoeia of People’s Republic of China was applied to determine the samples (UV-1100, Beijing Rayleigh Analytical Instrument Corporation) and the correlationship between two methods was showed as the following regression equation: CCL = 1.01CUV + 0.25

and

r 2 = 0.9995.

3.9.2. Determination of the Vitamin B12 in human serum According to the analytical procedure proposed under Section 2.5, the human serum samples were determined by proposed method with the standard addition method. And the experimental results samples were summarized in Table 2 and compared with the results obtained by radioimmunoassay (RIA) with a gamma counter (Cap RIA-16, Capintec Instruments, Inc.) in Radio-Immunity Center. The results obtained by the proposed CL method were well agree with that obtained by RIA, and the correlationship between two method was indicated as the following regression equation: CCL = 1.11CRIA − 0.04

and

r 2 = 0.9912.

3.9.3. Determination of Vitamin B12 in hen egg yolk and fish tissue Following the Section 2.6, Vitamin B12 , abundant in hen egg yolk, fish muscle and liver, were also determined by the proposed CL method. The contents of Vitamin B12 were (9.4 ± 1.1) × 10−8 g g−1 in hen

Table 1 Results of determination of Vitamin B12 in pharmaceutical preparationsa Sampleb

Added (×10−9 g l−1 )

Found (×10−9 g l−1 )

R.S.D. (%)

Recovery (%)

Content of Vitamin B12 (injection: g l−1 , tablets: 1.0 × 10−6 g per tablet) By proposed method

By UVc

Injection 1b

0 6.9

19.8 27.2

2.57 1.73

107.3

0.49 ± 0.01

0.47 ± 0.02

Injection 2b

0 13.8

20.4 34.0

2.54 1.85

98.0

0.51 ± 0.01

0.48 ± 0.01

Injection 3b

0 23.0

19.8 44.1

2.73 2.07

105.2

0.50 ± 0.02

0.47 ± 0.01

Tablets 1d

0 11.5

10.0 21.2

2.36 1.82

97.6

24.9 ± 0.7

24.7 ± 0.4

Tablets 2d

0 11.5

19.6 30.6

2.14 1.69

95.7

24.5 ± 0.6

24.4 ± 0.4

Tablets 3e

0 18.4

19.8 38.2

2.99 1.89

99.9

24.8 ± 0.5

24.2 ± 0.5

a

The average of five determinations. Batch nos. 200203154, 200209063 and 200108114, Jinxin Shuanghe Pharmaceutical Co. Ltd. c Results by reference method [27]. d Batch nos. 20021106 and 20020305, Pharmaceutical Plant of Puning, Chengde, Hebei Province. e Batch no. 20020704, Yunpeng Pharmaceutical Co. Ltd., Linfen, Shannxi Province. b

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77

Table 2 Results of determination of Vitamin B12 in human seruma Serum sample no.

Added (×10−9 g l−1 )

Found (×10−9 g l−1 )

R.S.D. (%)

1

0 0.30

0.36 0.64

4.62 3.68

2

0 0.50

0.35 0.87

3

0 1.00

4

Recovery (%)

Content of Vitamin B12 (×10−6 g l−1 ) By proposed method

By RIAb

92.0

0.366 ± 0.017

0.367 ± 0.030

4.22 3.33

103.3

0.350 ± 0.015

0.348 ± 0.031

0.28 1.36

4.22 3.46

107.8

0.284 ± 0.012

0.291 ± 0.033

0 0.40

0.42 0.80

4.65 3.16

94.2

0.423 ± 0.020

0.415 ± 0.025

5

0 0.50

0.39 0.92

4.08 3.83

107.0

0.390 ± 0.016

0.381 ± 0.029

6

0 1.00

0.39 1.42

4.26 3.18

102.8

0.389 ± 0.017

0.386 ± 0.026

7

0 0.40

0.40 0.82

4.88 3.84

95.0

0.403 ± 0.019

0.405 ± 0.022

8

0 0.50

0.39 0.92

4.08 3.83

106.3

0.389 ± 0.016

0.382 ± 0.027

9

0 1.00

0.31 1.38

4.47 3.21

106.8

0.312 ± 0.014

0.320 ± 0.030

a b

The average of five determinations. The results were supplied by Radio-Immunity Center, People’s Hospital of Shannxi Province.

egg yolk, (9.42 ± 0.16) × 10−8 g g−1 in fish liver and (0.51 ± 0.02) × 10−8 g g−1 in the fish muscle, respectively with the recoveries from 96.5 to 107.6% and R.S.D. of less than 3.0%. Furthermore, the results by proposed method were well agreed with the reference contents as 3.0 ∼ 10.0 × 10−8 g g−1 [32]. 3.9.4. Determination of the Vitamin B12 in spiked human serum and urine samples The proposed method was applied to determine Vitamin B12 in the spiked serum and urine samples. Vitamin B12 in six spiked serum samples and three urine samples were determined by the standard addition method. And the results for spiked serum were (74.4±0.4)×10−3 and (50.8±0.8)×10−3 g l−1 with recoveries from 94.9 to 103.3%. For spiked urine samples, the results were (1.01 ± 0.02) × 10−3 , (0.76 ± 0.01) × 10−3 and (0.48 ± 0.01) × 10−3 g l−1 with recoveries from 98.8 to 103.1%.

4. Possible CL mechanism of the reaction As the above description in Section 3.1, it was noticeable that carbonate, which was generated by dissolved CO2 in alkaline medium, inhibited the luminol–dissolved oxygen–Vitamin B12 CL signal. And then, the possible mechanism of the cobalt–luminol–dissolved oxygen CL reaction was discussed by two experiments. Firstly, the CL emission spectrum was examined using an RF-540 spectrofluorimeter, showing the maximum CL emission wavelength as 425 nm, which suggested that luminol was oxidized to excited 3-aminophthalate by • O − radicals in alkali medium and emits CL signals 2 [33–35]. Secondly, the CL intensities generated separately by online ultrasonically degassed solutions and general solutions were compared. With regard to the increment of the CL intensity, it decreased by 97.1% with the degassed solutions, which suggested that the dissolved oxygen reacts with cobalt(II) [32]. We

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suggested that this cobalt(II)–luminol–dissolved oxygen CL reaction could be described as following reactions:

ticals and determination of Vitamin B12 in biological samples.

1. Ionization of luminol in alkaline aqueous and formation of luminiol–cobalt(II)–hydroxy ion complex [36]. 2. Reaction of dissolved oxygen and the former complex to yield cobalt–oxygen complex.

References

HO–Co–LuH + O2 → + Co–O–O∗

(1)

3. Luminol anion reacted with cobalt-oxygen complex to produce cobalt(III) and • O2 − radicals, which was described as follows: OH

O

C*

N NH NH2

+

+

Co O

N

O*

N

O

NH2

O

O-

-

C*

+

N

H

+

N

+ H

NH 2

+

3+

Co

+

.O2

O

(2) 4. Oxidation of luminol radical anion by cobalt(III) to generate 3-aminoazaquione [37]. 5. Oxidation of 3-aminoazaquione by • O2 − radicals to yield 3-aminophthalate excited state for emission of light [33–35]. 5. Conclusions A sensitive and simple CL method has been proposed for sub-picogram determination of Vitamin B12 at in pharmaceuticals and human serum. Combined with the FI system, the enhancive cobalt(II)–luminol–dissolved oxygen CL was utilized for determination of Vitamin B12 . The presented method takes prominent advantages including instrumental simplicity, reduced reagents consumption, improved sensitivity, analytical efficiency, and easy handling procedure as well. The present method offers the promise for routine quality control of pharmaceu-

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